Wall Perturbation Research Articles

Functional characterization of BbEaf6 in Beauveria bassiana: Implications for fungal virulence and stress response

ABSTRACT The Eaf6 protein, a conserved component of the NuA4 and NuA3 complexes in yeast and MOZ/MORF complexes in humans, plays crucial roles in transcriptional activation, gene regulation, and cell cycle control. Despite its significance in other organisms, the functional role of Eaf6 in entomopathogenic fungi (EPF) remained unexplored. Here, we investigate the function of BbEaf6, the Eaf6 homolog in the entomopathogenic fungus Beauveria bassiana. We demonstrate that BbEaf6 is predominantly localized in nuclei, similar to its counterpart in other fungi. Deletion of BbEaf6 resulted in delayed conidiation, reduced conidial yield, and altered conidial properties. Transcriptomic analysis revealed dysregulation of the genes involved in asexual development and cell cycle progression in the ΔBbEaf6 mutant. Furthermore, the ΔBbEaf6 mutant exhibited decreased tolerance to various stresses, including ionic stress, cell wall perturbation, and DNA damage stress. Notably, the ΔBbEaf6 mutant displayed attenuated virulence in insect bioassays, accompanied by dysregulation of genes associated with cuticle penetration and haemocoel infection. Overall, our study elucidates the multifaceted role of BbEaf6 in stress response, development, and virulence in B. bassiana, providing valuable insights into the molecular mechanisms governing fungal pathogenesis and potential targets for pest management strategies.

Glyoxal oxidase-mediated detoxification of reactive carbonyl species contributes to virulence, stress tolerance, and development in a pathogenic fungus.

Reactive carbonyl and oxygen species (RCS/ROS), often generated as metabolic byproducts, particularly under conditions of pathology, can cause direct damage to proteins, lipids, and nucleic acids. Glyoxal oxidases (Gloxs) oxidize aldehydes to carboxylic acids, generating hydrogen peroxide (H2O2). Although best characterized for their roles in lignin degradation, Glox in plant fungal pathogens are known to contribute to virulence, however, the mechanism underlying such effects are unclear. Here, we show that Glox in the insect pathogenic fungus, Metarhizium acridum, is highly expressed in mycelia and during formation of infection structures (appressoria), with the enzyme localizing to the cell membrane. MaGlox targeted gene disruption mutants showed RCS and ROS accumulation, resulting in cell toxicity, induction of apoptosis and increased autophagy, inhibiting normal fungal growth and development. The ability of the MaGlox mutant to scavenge RCS was significantly reduced, and the mutant exhibited increased susceptibility to aldehydes, oxidative and cell wall perturbing agents but not toward osmotic stress, with altered cell wall contents. The ΔMaGlox mutant was impaired in its ability to penetrate the host cuticle and evade host immune defense resulting in attenuated pathogenicity. Overexpression of MaGlox promoted fungal growth and conidial germination, increased tolerance to H2O2, but had little to other phenotypic effects. Transcriptomic analyses revealed downregulation of genes related to cell wall synthesis, conidiation, stress tolerance, and host cuticle penetration in the ΔMaGlox mutant. These findings demonstrate that MaGlox-mediated scavenging of RCS is required for virulence, and contributes to normal fungal growth and development, stress resistance.

Genome-Wide Identification of SNARE Family Genes and Functional Characterization of an R-SNARE Gene BbSEC22 in a Fungal Insect Pathogen Beauveria bassiana.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central components of the machinery mediating cell membrane fusion and intracellular vesicular trafficking in eukaryotic cells, and have been well-documented to play critical roles in growth, development, and pathogenesis in the filamentous fungal plant pathogens. However, little is known about the contributions of SNAREs to the physiology and biocontrol potential in entomopathogenic filamentous fungi. Here, a genome-wide analysis of SNARE genes was performed taking advantage of the available whole genome sequence of Beauveria bassiana, a classical entomopathogenic fungus. Based on the compared genomic method, 22 genes encoding putative SNAREs were identified from the whole genome of B. bassiana, and were classified into four groups (7 Qa-, 4 Qb-, 6 Qc-, and 5 R-SNAREs) according to the conserved structural features of their encoding proteins. An R-SNARE encoding gene BbSEC22 was further functionally characterized by gene disruption and complementation. The BbSEC22 null mutant showed a fluffy appearance in mycelial growth and an obvious lag in conidial germination. The null mutant also exhibited significantly increased sensitivity to oxidative stress and cell wall perturbing agents and reduced the yield of conidia production by 43.1% compared with the wild-type strain. Moreover, disruption of BbSEC22 caused a significant decrease in conidial virulence to Spodoptera litura larvae. Overall, our results provide an overview of vesicle trafficking in B. bassiana and revealed that BbSec22 was a multifunctional protein associated with mycelial growth, sporulation, conidial germination, stress tolerance, and insecticidal virulence.

Unveiling the molecular networks underlying cellular impairment in Saccharomyces cerevisiae: investigating the effects of magnesium oxide nanoparticles on cell wall integrity and endoplasmic reticulum stress response.

Nanoparticles, particularly magnesium oxide nanoparticles (MgO-NPs), are increasingly utilized in various fields, yet their potential impact on cellular systems remains a topic of concern. This study aimed to comprehensively investigate the molecular mechanisms underlying MgO-NP-induced cellular impairment in Saccharomyces cerevisiae, with a focus on cell wall integrity, endoplasmic reticulum (ER) stress response, mitochondrial function, lipid metabolism, autophagy, and epigenetic alterations. MgO-NPs were synthesized through a chemical reduction method, characterized for morphology, size distribution, and elemental composition. Concentration-dependent toxicity assays were conducted to evaluate the inhibitory effect on yeast growth, accompanied by propidium iodide (PI) staining to assess membrane damage. Intracellular reactive oxygen species (ROS) accumulation was measured, and chitin synthesis, indicative of cell wall perturbation, was examined along with the expression of chitin synthesis genes. Mitochondrial function was assessed through Psd1 localization, and ER structure was analyzed using dsRed-HDEL marker. The unfolded protein response (UPR) pathway activation was monitored, and lipid droplet formation and autophagy induction were investigated. Results demonstrated a dose-dependent inhibition of yeast growth by MgO-NPs, with concomitant membrane damage and ROS accumulation. Cell wall perturbation was evidenced by increased chitin synthesis and upregulation of chitin synthesis genes. MgO-NPs impaired mitochondrial function, disrupted ER structure, and activated the UPR pathway. Lipid droplet formation and autophagy were induced, indicating cellular stress responses. Additionally, MgO-NPs exhibited differential cytotoxicity on histone mutant strains, implicating specific histone residues in cellular response to nanoparticle stress. Immunoblotting revealed alterations in histone posttranslational modifications, particularly enhanced methylation of H3K4me. This study provides comprehensive insights into the multifaceted effects of MgO-NPs on S. cerevisiae, elucidating key molecular pathways involved in nanoparticle-induced cellular impairment. Understanding these mechanisms is crucial for assessing nanoparticle toxicity and developing strategies for safer nanoparticle applications.

FpOGT is required for fungal growth, stress response, and virulence of Fusarium proliferatum by affecting the expression of glucokinase and other glucose metabolism-related genes

O-GlcNAcylation, an important post-translational modification catalyzed by O-GlcNAc transferase (OGT), plays critical roles in several biological processes. In this study, we present our findings on the function of FpOGT in regulating physiological processes and pathogenicity of Fusarium proliferatum (Fp), the alfalfa root rot fungus. The deletion of FpOGT impaired mycelial growth and altered macroconidia morphology in Fp. Furthermore, ΔFpOGT mutant displayed altered tolerance to various stressors, including cell wall perturbing agents, osmotic stressors, metal ionic stressors, and fungicides. Deletion of FpOGT significantly decreased Fp virulence toward alfalfa. The transcriptome analysis demonstrated that FpOGT plays a regulatory role in glucose metabolic pathways, including glycolysis, tricarboxylic acid (TCA) cycle, and hexosamine biosynthesis pathway (HBP), by influencing the expression of relevant genes. The downregulation of the glucokinase gene, FpGCK, was observed in ΔFpOGT, and the disruption of FpGCK led to a decrease in Fp virulence. Additionally, FpOGT affected the expression levels of the FpGCK-AS1 isoform, thereby impacting glucokinase function. The molecular docking analysis elucidated the plausible physical interaction between FpOGT and FpGCK, thereby offering valuable insights into their interrelationship. These findings underscore the indispensable involvement of FpOGT, the sole O-GlcNAc transferase in Fp, in various biological processes and the pathogenicity through its regulation of fundamental metabolic processes. Consequently, this study emphasizes the significance and elucidates the molecular mechanism underlying the role of O-GlcNAc transferase in diverse fundamental biological processes and the pathogenicity of phytopathogenic fungi.

Analysis of Slip Effects on the Stability and Interactions of Mach 5 Flat-Plate Boundary-Layer Waves

The issue of boundary-layer transition with slight rarefaction for high-speed aircraft has recently received increased attention. In this paper, the stability and resonant interactions of boundary-layer waves for a Mach 5 flat plate under the slip effect are investigated using direct numerical simulation (DNS) and linear stability theory (LST). The slip effect is modelled by slip boundary conditions within the Navier-Stokes (NS) framework, and an LST method considering wall perturbations for both no-slip and slip flows is established. The present numerical results indicate that the slip effect has a consistent impact on the stability of fast and slow modes as obtained from DNS and current LST. In addition, the DNS results show that the slip effect delays the conversion of mode F1 to mode S led by their resonance, and enhances the resonant interactions between fast mode waves and fast acoustic waves.

Sterol interactions influence the function of Wsc sensors

The Wsc1, Wsc2, and Wsc3 proteins are essential cell surface sensors that respond to cell wall perturbation by activating the cell wall integrity pathway (CWIP). We show here that in situ production of cholesterol (in place of ergosterol) induces hyper-phosphorylation of Slt2, the MAPK of the CWIP, and upregulates cell wall biosynthesis. Deletion of all three Wsc genes in K. phaffii reverts these phenotypes. In the cholesterol-producing strain, both Wsc1 and Wsc3 accumulate in the plasma membrane. Close inspection of the transmembrane domains of all three Wsc proteins predicted by AlphaFold2 revealed the presence of CRAC sterol-binding motifs. Experiments using a photoreactive cholesterol derivative indicate intimate interaction of this sterol with the Wsc transmembrane domain, and this apparent sterol binding was abrogated in Wsc mutants with substitutions in the CRAC motif. We also observed cholesterol interaction with CRAC-like motifs in the transmembrane domains of mammalian integrins, analogs of Wsc proteins. Our results suggest that proper signaling of the Wsc sensors requires highly specific binding of the native endogenous terminal sterol, ergosterol.

Impact of PpSpi1, a glycosylphosphatidylinositol-anchored cell wall glycoprotein, on cell wall defects of N-glycosylation-engineered Pichia pastoris.

Engineering of biological pathways in various microorganisms is a promising direction for biotechnology. Since the existing microbial cells have evolved over a long period of time, any artificial engineering may cause some unexpected and harmful effects on them. Systematically studying and evaluating these engineered strains are very important and necessary. In order to produce therapeutic proteins with human-like N-glycan structures, much progress has been achieved toward the humanization of N-glycosylation pathways in yeasts. The properties of a P. pastoris strain with humanized N-glycosylation machinery were carefully evaluated in this study. Our work has identified a key glycoprotein (PpSpi1) responsible for the poor growth and morphological defects of this glycoengineered strain. Overexpression of PpSpi1 could significantly rescue the growth defect of the glycoengineered P. pastoris and facilitate its future industrial applications.

Deubiquitination module is critical for oxidative stress response and biofilm formation in Candida glabrata.

Candidiasis is one of the most important fungal diseases and generally refers to diseases of the skin or mucosal tissues caused by Candida species. Candida glabrata is an opportunistic human fungal pathogen. Infection with C. glabrata has significantly increased due to innate antifungal drug tolerance and the ability to adhere to mucocutaneous surfaces. Spt-Ada-Gcn5 acetyltransferase complex contains two different post-translational modifications, histone acetylation (HAT) module and deubiquitination (DUB) module, which are decisive in gene regulation and highly conserved in eukaryotes. Previous research in our laboratory found that the HAT module ADA2 could regulate C. glabrata oxidative stress tolerance, drug tolerance, cell wall integrity, and virulence. However, the roles of the DUB module that is comprised of UBP8, SGF11, SGF73, and SUS1 genes in those phenotypes are not yet understood. In this study, we found that DUB module genes UBP8, SGF11, and SUS1, but not SGF73 positively regulate histone H2B DUB. Furthermore, ubp8, sgf11, and sus1 mutants exhibited decreased biofilm formation and sensitivity to cell wall-perturbing agent sodium dodecyl sulfate and antifungal drug amphotericin B. In addition, the sgf73 mutant showed increased biofilm formation but was susceptible to oxidative stresses, antifungal drugs, and cell wall perturbing agents. The ubp8, sgf11, and sus1 mutants showed marginal hypovirulence, whereas the sgf73 mutant exhibited virulence similar to the wild type in a murine systemic infection model. In conclusion, the C. glabrata DUB module plays distinct roles in H2B ubiquitination, oxidative stress response, biofilm formation, cell wall integrity, and drug tolerance, but exhibits minor roles in virulence.

Iron and copper on Botrytis cinerea: new inputs in the cellular characterization of their inhibitory effect.

Certain metals play key roles in infection by the gray mold fungus, Botrytis cinerea. Among them, copper and iron are necessary for redox and catalytic activity of enzymes and metalloproteins, but at high concentrations they are toxic. Understanding the mechanism requires more cell characterization studies for developing new, targeted metal-based fungicides to control fungal diseases on food crops. This study aims to characterize the inhibitory effect of copper and iron on B. cinerea by evaluating mycelial growth, sensitivity to cell wall perturbing agents (congo red and calcofluor white), membrane integrity, adhesion, conidial germination, and virulence. Tests of copper over the range of 2 to 8 mM and iron at 2 to 20 mM revealed that the concentration capable of reducing mycelial growth by 50% (IC50) was 2.87 mM and 9.08 mM for copper and iron, respectively. When mixed at equimolar amounts there was a significant inhibitory effect mostly attributable to copper. The effect of Cu50, Fe50, and Cu50-Fe50 was also studied on the mycelial growth of three wild B. cinerea strains, which were more sensitive to metallic inhibitors. A significant inhibition of conidial germination was correlated with adhesion capacity, indicating potential usefulness in controlling disease at early stages of crop growth. Comparisons of the effects of disruptive agents on the cell wall showed that Cu, Fe, and Cu-Fe did not exert their antifungal effect on the cell wall of B. cinerea. However, a relevant effect was observed on plasma membrane integrity. The pathogenicity test confirmed that virulence was correlated with the individual presence of Cu and Fe. Our results represent an important contribution that could be used to formulate and test metal-based fungicides targeted at early prevention or control of B. cinerea.

Chitin synthases containing myosin motor-like domain are required for cell wall integrity and virulence of vascular wilt pathogen Verticillium dahliae

Verticillium wilt (VW) of cotton poses a serious threat to the quality and yield of cotton. Verticillium dahliae is the primary causal agent of cotton VW. Moreover, V. dahliae can infect more than 200 species of dicotyledonous plants. The fungal cell wall plays a crucial role in its growth, development and pathogenicity. However, the mechanism of cell wall synthesis in V. dahliae and its role in pathogenesis remains unclear. In this study, we identified two chitin synthase (CHS) genes VdChs5 and VdChs7 containing myosin motor-like domain (MMD) and characterized their role in virulence of V. dahliae. The results showed that the functions of VdChs5 and VdChs7 were largely redundant, and target deletion of both VdChs5 and VdChs7 in V. dahliae did not affect vegetative growth, but reduced conidial production. ΔVdChs5Chs7 deletion mutant failed to colonize and proliferate in cotton vascular tissue, and exhibited significantly reduced virulence on cotton, suggesting that VdChs5 and VdChs7 are necessary for pathogenesis. In addition, the thickness of the cell wall in ΔVdChs5Chs7 showed significantly decreased, and ΔVdChs5Chs7 mutant exhibited hypersensitivity to cell wall perturbing agents and reactive oxygen species (ROS), indicating that VdChs5 and VdChs7 play key roles in cell wall integrity. Further, host-induced gene silencing (HIGS) silenced transcripts of VdChs5 and VdChs7 in susceptible cotton (Gossypium hirsutum L. acc. TM-1) enhanced resistance to cotton VW. Taken together, our data demonstrated that VdChs5 and VdChs7 play pivotal roles in proliferation, cell wall integrity, and pathogenicity, and provided a novel strategy to improve Verticillium wilt resistance in cotton and other susceptible host plants.

Tetracarboxylic acid transporter regulates growth, conidiation, and carbon utilization in Metarhizium acridum.

Carbon sources and their utilization are vital for fungal growth and development. C4-dicarboxylic acids are important carbon and energy sources that function as intermediate products of the tricarboxylic acid cycle. Transport and regulation of C4-dicarboxylic acid uptake are mainly dependent on tetracarboxylic acid transporters (Dcts) in many microbes, although the roles of Dct genes in fungi have only been partially characterized. Here, we report on the functions of two Dct genes (Dct1 and Dct2) in the entomopathogenic fungus Metarhizium acridum. Our data showed that loss of the MaDct1 gene affected utilization of tetracarboxylic acids and other carbon sources. ΔMaDct1 mutants showed larger colony sizes with extensive mycelial growth but were delayed in conidiation with decreased conidia yield as compared to the wild-type parental strain. On the nutrient-deficient medium, SYA, the wild-type strain produced microcycle conidia, whereas the ΔMaDct1 mutant produced (normal) aerial conidia. In addition, ΔMaDct1 had decreased tolerance to cell wall perturbing agents, but increased tolerances to UV-B radiation and osmotic stress. Insect bioassays indicated that loss of MaDct1 did not affect pathogenicity. In contrast, no distinct phenotypic change was observed for the MaDct2 mutant in terms of growth and biocontrol characteristics. Transcriptomic profiling between wild type and ΔMaDct1 showed that differentially expressed genes were enriched in carbohydrate and amino acid metabolism, transport and catabolism, and signal transduction. These results demonstrate that MaDct1 regulates the conidiation pattern shift and mycelial growth by affecting utilization of carbon sources. These findings are helpful for better understanding the effect of intermediates of carbon metabolism on fungal growth and conidiation. KEY POINTS: • MaDct1 influences fungal growth and conidiation by affecting carbon source utilization. • MaDct1 regulates conidiation pattern shift under nutrient deficiency condition. • MaDct1 is involved in stress tolerance and has no effect on virulence. • MaDct2 has no effect on growth and biocontrol characteristic.

An Easy Method to Determine the Effective Conductivity of Carbon Fiber Composites Using a Wall Perturbation Approach

Carbon-fiber-reinforced plastics (CFRPs) are of increasing popularity in a wide range of applications, and microwave curing promises significant reduction in processing times. However, for the design of an efficient microwave curing system, the composites' effective material parameters must be known. This work presents a measurement system using a wall perturbation approach with a coaxial cavity to determine the effective conductivity of a CFRP along the fiber direction.

The Gcn5-Ada2-Ada3 histone acetyltransferase module has divergent roles in pathogenesis of Candida glabrata.

Candida glabrata is an opportunistic fungal pathogen and the second most prevalent species isolated from candidiasis patients. C. glabrata has intrinsic tolerance to antifungal drugs and oxidative stresses and the ability to adhere to mucocutaneous surfaces. However, knowledge about the regulation of its virulence traits is limited. The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex modulates gene transcription by histone acetylation through the histone acetyltransferase (HAT) module comprised of Gcn5-Ada2-Ada3. Previously, we showed that the ada2 mutant was hypervirulent but displayed decreased tolerance to antifungal drugs and cell wall perturbing agents. In this study, we further characterized the functions of Ada3 and Gcn5 in C. glabrata. We found that single, double, or triple deletions of the HAT module, as expected, resulted in a decreased level of acetylation on histone H3 lysine 9 (H3K9) and defective growth. These mutants were more susceptible to antifungal drugs, oxidative stresses, and cell wall perturbing agents compared with the wild-type. In addition, HAT module mutants exhibited enhanced agar invasion and upregulation of adhesin and proteases encoding genes, whereas the biofilm formation of those mutants was impaired. Interestingly, HAT module mutants exhibited enhanced induction of catalases (CTA1) expression upon treatment with H2O2 compared with the wild-type. Lastly, although ada3 and gcn5 exhibited marginal hypervirulence, the HAT double and triple mutants were hypervirulent in a murine model of candidiasis. In conclusion, the HAT module of the SAGA complex plays unique roles in H3K9 acetylation, drug tolerance, oxidative stress response, adherence, and virulence in C. glabrata.

Plant Cell Wall Integrity Perturbations and Priming for Defense.

A plant cell wall is a highly complex structure consisting of networks of polysaccharides, proteins, and polyphenols that dynamically change during growth and development in various tissues. The cell wall not only acts as a physical barrier but also dynamically responds to disturbances caused by biotic and abiotic stresses. Plants have well-established surveillance mechanisms to detect any cell wall perturbations. Specific immune signaling pathways are triggered to contrast biotic or abiotic forces, including cascades dedicated to reinforcing the cell wall structure. This review summarizes the recent developments in molecular mechanisms underlying maintenance of cell wall integrity in plant-pathogen and parasitic interactions. Subjects such as the effect of altered expression of endogenous plant cell-wall-related genes or apoplastic expression of microbial cell-wall-modifying enzymes on cell wall integrity are covered. Targeted genetic modifications as a tool to study the potential of cell wall elicitors, priming of signaling pathways, and the outcome of disease resistance phenotypes are also discussed. The prime importance of understanding the intricate details and complete picture of plant immunity emerges, ultimately to engineer new strategies to improve crop productivity and sustainability.

Comparative Transcriptome Analysis Identifies Key Defense Genes and Mechanisms in Mulberry (Morus alba) Leaves against Silkworms (Bombyx mori).

As a consequence of long-term coevolution and natural selection, the leaves of mulberry (Morus alba) trees have become the best food source for silkworms (Bombyx mori). Nevertheless, the molecular and genomic basis of defense response remains largely unexplored. In the present study, we assessed changes in the transcriptome changes of mulberry in response to silkworm larval feeding at 0, 3, and 6 h. A total of 4709 (up = 2971, down = 1738) and 3009 (up = 1868, down = 1141) unigenes were identified after 3 and 6 h of silkworm infestation, respectively. MapMan enrichment analysis results show structural traits such as leaf surface wax, cell wall thickness and lignification form the first physical barrier to feeding by the silkworms. Cluster analysis revealed six unique temporal patterns of transcriptome changes. We predicted that mulberry promoted rapid changes in signaling and other regulatory processes to deal with mechanical damage, photosynthesis impairment, and other injury caused by herbivores within 3-6 h. LRR-RK coding genes (THE1, FER) was predicted participated in perception of cell wall perturbation in mulberry responding to silkworm feeding. Ca2+ signal sensors (CMLs), ROS (OST1, SOS3), RBOHD/F, CDPKs, and ABA were part of the regulatory network after silkworm feeding. Jasmonic acid (JA) signal transduction was predicted to act in silkworm feeding response, 10 JA signaling genes (such as OPR3, JAR1, and JAZ1) and 21 JA synthesis genes (such as LOX2, AOS, and ACX1) were upregulated after silkworm feeding for 3 h. Besides, genes of "alpha-Linolenic acid metabolism" and "phenylpropanoid biosynthesis" were activated in 3 h to reprogram secondary metabolism. Collectively, these findings provided valuable insights into silkworm herbivory-induced regulatory and metabolic processes in mulberry, which might help improve the coevolution of silkworm and mulberry.

Amphiphysin AoRvs167-Mediated Membrane Curvature Facilitates Trap Formation, Endocytosis, and Stress Resistance in Arthrobotrys oligospora.

Bin1/Amphiphysin/Rvs (BAR) domain-containing proteins mediate fundamental cellular processes, including membrane remodeling and endocytosis. Nematode-trapping (NT) fungi can differentiate to form trapping structures through highly reorganized cell membranes and walls. In this study, we identified the NT fungus Arthrobotrys oligospora ortholog of yeast Rvs167 and documented its involvement in membrane bending and endocytosis. We further confirmed that the deletion of AoRvs167 makes the fungus more hypersensitive to osmotic salt (Nacl), higher temperatures (28 to 30 °C), and the cell wall perturbation agent Congo red. In addition, the disruption of AoRvs167 reduced the trap formation capacity. Hence, AoRvs167 may regulate fungal pathogenicity through the integrity of plasma membranes and cell walls.

Covalent Organic Frameworks: Chemistry of Pore Interface and Wall Surface Perturbation and Impact on Functions

ConspectusPorous structures are ubiquitous, discovered in nature ranging from biological channels in animals and plants to various pores in sediments and minerals and playing vital roles in maintaining biological activities and ecosystems. Synthetic pores have been known for over 100 years and currently continue to be a central subject in the fields of chemistry, physics, materials science, and technology. A fundamental key issue is how to develop specific functions with pores. Pores are determined by three parameters, including pore shape, size, and environment; how to design these parameters in a controlled manner is a key subject. Covalent organic frameworks (COFs) are a distinct class of crystalline porous polymers as they combine covalent and noncovalent chemistries to result in long-range-ordered polygonal skeletons and discrete pores. The topological diagram─the principle for designing COFs─enables the predesign of not only skeletons but also pores, offering a powerful molecular platform for constructing tailor-made organic/polymeric materials. Over the past decade, progress in chemistry has greatly enhanced our capability to synthesize COFs to achieve different structures. Especially, the pores in COFs are constructed with lightweight elements, covalent bonds, and organic components, which offer numerous combinations to design and synthesize pore shapes, sizes, and interfaces. These parameters control the interplay with guest molecules and ions to determine the property and function of pores. Among various synthetic porous materials, COFs are unique in that these pore parameters are topologically designable and synthetically controllable. Two complementary strategies, i.e., topology diagram and pore surface engineering, have been developed for pore chemistry, in which the first one emphasizes the in situ approach to design and control pores and the second one highlights the postsynthetic way to tune the pore structures finely yet precisely. These two different chemistries offer individual ways to explore different pores, properties, and functional materials and systems. Looking at the features of COFs, a basic common structure of the pores is the well-defined pore interface, which is constituted by aligned surface atoms and side units at a proximate distance along the pore long-axial direction and distributed periodically over the pores. These features of pore interfaces are specific to COFs and predetermine their functions. In this Account, we scrutinize the chemistry of pore interfaces by focusing on the pore shape, pore size, and aligned atoms and units on pore walls to design unique properties and functions. We highlight pore wall perturbation with surface atoms and side units to demonstrate their decisive roles in structural formation and functional expression. We summarize perspectives, key issues to be addressed, and opportunities with the aim of showing a promising way to next-stage materials and functional designs.

β-Nitrostyrene derivatives as broad range potential antifungal agents targeting fungal cell wall

The prevalence of multidrug resistance has been increasingly witnessed during the past few decades. Resistance of human pathogenic fungi against the currently available antifungal agents has increased the frequency of fungal infections and associated mortality rates. The discovery of novel lead antifungal agents is important to challenge multidrug resistance. The present study examined the antifungal potential of chemically synthesized β-Nitrostyrene derivatives. Among the eight β-Nitrostyrene derivatives used in this study, SS45, SS46 and SS47 showed strong antifungal potential. The results show that β-Nitrostyrene derivatives inhibited the growth of different species of human pathogenic Candida, particularly the highly prevalent C. albicans, C. glabrata and the emerging pathogenic C. auris species. Moreover, β-Nitrostyrene derivatives also show strong antifungal activities against drug-resistant clinical isolates and drug transporter overexpressing fungal species. The drug susceptibility assays revealed that β-Nitrostyrene derivatives are fungicidal and show the synergy of action when combined with antifungal drugs caspofungin and fluconazole. The transcriptomic study performed on C. albicans in the presence of β-Nitrostyrene derivatives revealed the differential expression of genes related to cell wall metabolism. Mechanistically, β-Nitrostyrene derivatives impact cell wall morphology, enhance ROS generation and modulate drug efflux. Collectively this study reveals that β-Nitrostyrene derivatives have strong antifungal potential with a particular mode of activity similar to known cell wall perturbing antifungal agents and thus can be exploited as promising potential antifungal agents for further studies.

Comparative Transcriptomics Reveal Possible Mechanisms of Amphotericin B Resistance in Candida auris.

Candida auris is an emerging multidrug-resistant human fungal pathogen often refractory to treatment by all classes of antifungal drugs. Amphotericin B (AmB) is a fungicidal drug that, despite its toxic side effects, remains a drug of choice for the treatment of drug-resistant fungal infections, including those caused by C. auris. However, the molecular mechanisms underlying AmB resistance are poorly understood. In this study, we present data that suggests membrane lipid alterations and chromatin modifications are critical processes that may contribute to or cause adaptive AmB resistance in clinical C. auris isolates. To determine the plausible cause of increased AmB resistance, we performed RNA-seq of AmB-resistant and sensitive C. auris isolates. Remarkably, AmB-resistant strains show a pronounced enrichment of genes involved in lipid and ergosterol biosynthesis, adhesion, drug transport as well as chromatin remodeling. The transcriptomics data confirm increased adhesion and reduced lipid membrane permeability of AmB-resistant strains compared to the sensitive isolates. The AmB-resistant strains also display hyper-resistance to cell wall perturbing agents, including Congo red, calcofluor white and caffeine. Additionally, we noticed an increased phosphorylation of Mkc1 cell integrity MAP kinase upon AmB treatment. Collectively, these data identify differences in the transcriptional landscapes of AmB-resistant versus AmB-sensitive isolates and provide a framework for the mechanistic understanding of AmB resistance in C. auris.