BnXTH1 regulates cadmium tolerance by modulating vacuolar compartmentalization and the cadmium binding capacity of cell walls in ramie (Boehmeria nivea).

Xyloglucan endotransglucosylase/hydrolases (XTH) are cell wall-modifying enzymes important in plant response to abiotic stress. However, the role of XTH in cadmium (Cd) tolerance in ramie remains largely unknown. Here, we identified and cloned BnXTH1, a member of the XTH family, in response to Cd stress in ramie. The BnXTH1 promoter (BnXTH1p) demonstrated that MeJA induces the response of BnXTH1p to Cd stress. Moreover, overexpressing BnXTH1 in Boehmeria nivea increased Cd tolerance by significantly increasing the Cd content in the cell wall and decreasing Cd inside ramie cells. Cadmium stress induced BnXTH1-expression and consequently increased xyloglucan endotransglucosylase (XET) activity, leading to high xyloglucan contents and increased hemicellulose contents in ramie. The elevated hemicellulose content increased Cd chelation onto the cell walls and reduced the level of intracellular Cd. Interestingly, overexpressing BnXTH1 significantly increased the content of Cd in vacuoles of ramie and vacuolar compartmentalization genes. Altogether, these results evidence that Cd stress induced MeJA accumulation in ramie, thus, activating BnXTH1 expression and increasing the content of xyloglucan to enhance the hemicellulose binding capacity and increase Cd chelation onto cell walls. BnXTH1 also enhances the vacuolar Cd compartmentalization and reduces the level of Cd entering the organelles and soluble solution.

Encapsulation of carvacrol and thymol with yeast cell wall and its repellent activity against Amblyomma sculptum and Rhipicephalus sanguineus (Sensu Lato).

The main way to avoid contact with ticks and consequently tick-borne disease is the use of synthetic repellents. The search of new repellent compounds to increase the possibilities of use in strategies controls are necessary. The present study evaluated the repellent activity of two natural terpenes carvacrol and thymol in each one two different formulation (encapsulated and nonencapsulated with yeast cell wall) against the ticks Amblyomma sculptum and Rhipicephalus sanguineus sensu lato nymphs. Nymphs of A. sculptum and R. sanguineus s.l. of a single generation were used. The vertical filter paper repellency assay were performed with different concentration of both terpenes encapsulated and nonencapsulated in yeast cell wall. The repellent concentration 50% (RC50) were calculated to each compound formulation. Both carvacrol and thymol (encapsulated and nonencapsulated), had a repellent activity against A. sculptum and R. sanguineus s.l nymphs. Amblyomma sculptum was more sensitive to nonencapsulated carvacrol (RC50 values: 0.0032 to 0.0082 mg/cm2 after 1 and 15 min) (P < 0.05), while R. sanguineus s.l. was more sensitive to encapsulated carvacrol (RC50 values: 0.00008 to 0.0035 mg/cm2 after 1 and 15 min) (P < 0.05). Among tick species, R. sanguineus s.l. was more sensitive for most compounds than A. sculptum (P < 0.05). Although with distinct repellent activities, carvacrol and thymol encapsulated can be a promising alternative to synthetic repellents against A. sculptum and R. sanguineus s.l.

Ex-chitin-g news on drug-induced fungal epitope unmasking.

The microbial cell wall is an essential cellular organelle commonly targeted by antimicrobials. It is also a battleground of innate immune recognition where microbes can evade immune recognition by masking essential cell wall components. A recent study (A. S. Wagner, S. W. Lumsdaine, M. M. Mangrum, and T. B. Reynolds, mBio https://doi.org/10.1128/mbio.00074-23, 2023) provides insight into how echinocandin antifungals cause exposure of proinflammatory ß(1,3)-glucan by driving excess chitin production in the weakened cell wall. Although many environmental and biological activities perturb cell wall integrity and regulate ß(1,3)-glucan exposure, we still know little about which intracellular signaling components regulate the cell wall changes that result in disrupted cell wall architecture. Wagner et al. showed that calcineurin and the Mkc1p kinase regulate chitin deposition and ß(1,3)-glucan unmasking. They further identified chitin synthesis as a key driving force in cell wall structure disruption leading to epitope exposure. Their findings highlight how fungal cell wall dynamics have important implications for antifungal immunity and future drug development.

Mecanismos de expressão de resistência aos antibióticos e saúde pública / Mechanisms of expression of resistance to antibiotics and public health / Mecanismos de expresión de la resistencia a los antibióticos y salud pública

Com o avanço da medicina e o aumento do uso de antimicrobianos, a resistência microbiana vem se tornando um problema sério na saúde pública. Para que uma bactéria se torne resistente, são necessários vários fatores, entre eles, o uso indiscriminado e prolongado de antimicrobianos e as resistências intrínsecas e adquiridas. Nesse contexto, o objetivo do trabalho foi explorar os mecanismos de ação dos antimicrobianos, de resistência e a sua importância na saúde pública. Foram utilizadas para a presente pesquisa, as bases de dados Pubmed, Google acadêmico e Scielo. Segundo a Organização Mundial da Saúde define-se resistência ao antibiótico quando o mesmo não produz mais efeito. A inserção cada vez mais frequente de antimicrobianos favorece a resistência, onde provocam uma pressão seletiva sobre os microrganismos, tornando-os resistentes a diversas drogas. O uso indiscriminado de antimicrobianos é o principal fator de resistência microbiana, assim como o uso de antimicrobianos sem exame de cultura e teste de sensibilidade. Neste sentido, conclui-se que é de suma importância a atualização de protocolos que contenham os mecanismos de resistência bacteriana a fim de minimizar o uso indiscriminado de antimicrobianos, assim como capacitar os profissionais da saúde para este problema na saúde pública.

With the advance of medicine and the increase in the use of antimicrobials, microbial resistance has become a serious problem in public health. For a bacterium to become resistant, several factors are necessary, among them, the indiscriminate and prolonged use of antimicrobials and the intrinsic and acquired resistance. In this context, the objective of the work was to explore the mechanisms of action of antimicrobials, resistance and their importance in public health. Pubmed, Google academic and Scielo databases were used for this research. According to the World Health Organization, resistance to antibiotics is defined when it no longer has an effect. The increasingly frequent insertion of antimicrobials favors resistance, where they put selective pressure on microorganisms, making them resistant to various drugs. The indiscriminate use of antimicrobials is the main factor of microbial resistance, as well as the use of antimicrobials without culture examination and sensitivity test. In this sense, it is concluded that it is extremely important to update protocols that contain the mechanisms of bacterial resistance in order to minimize the indiscriminate use of antimicrobials, as well as to train health professionals for this problem in public health.

Con los avances de la medicina y el mayor uso de antimicrobianos, la resistencia microbiana se ha convertido en un grave problema de salud pública. Para que una bacteria se vuelva resistente son necesarios varios factores, entre ellos, el uso indiscriminado y prolongado de antimicrobianos y la resistencia intrínseca y adquirida. En este contexto, el objetivo de este trabajo fue explorar los mecanismos de acción de los antimicrobianos, la resistencia y su importancia en la salud pública. Para esta investigación se utilizaron las bases de datos Pubmed, Google Scholar y Scielo. Según la Organización Mundial de la Salud, la resistencia a un antibiótico se define cuando deja de producir efecto. El uso cada vez más frecuente de antimicrobianos favorece la resistencia, ya que provocan una presión selectiva sobre los microorganismos, haciéndolos resistentes a varios fármacos. El uso indiscriminado de antimicrobianos es el principal factor de resistencia microbiana, así como el uso de antimicrobianos sin pruebas de cultivo y sensibilidad. En este sentido, se concluye que es de suma importancia actualizar los protocolos que contienen los mecanismos de resistencia bacteriana para minimizar el uso indiscriminado de antimicrobianos, así como capacitar a los profesionales de la salud para este problema en la salud pública.

Teixobactin kills bacteria by a two-pronged attack on the cell envelope.

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1-3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a ß-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates.

Expression of a novel mycobacterial phosphodiesterase successfully lowers cAMP levels resulting in reduced tolerance to cell wall-targeting antimicrobials.

cAMP and antimicrobial susceptibility in mycobacteriaAntimicrobial tolerance, the ability to survive exposure to antimicrobials via transient nonspecific means, promotes the development of antimicrobial resistance (AMR). The study of the molecular mechanisms that result in antimicrobial tolerance is therefore essential for the understanding of AMR. In gram-negative bacteria, the second messenger molecule 3'',5''-cAMP has been previously shown to be involved in AMR. In mycobacteria, however, the role of cAMP in antimicrobial tolerance has been difficult to probe due to its particular complexity. In order to address this difficulty, here, through unbiased biochemical approaches consisting in the fractionation of clear protein lysate from a mycobacterial strain deleted for the known cAMP phosphodiesterase (Rv0805c) combined with mass spectrometry techniques, we identified a novel cyclic nucleotide-degrading phosphodiesterase enzyme (Rv1339) and developed a system to significantly decrease intracellular cAMP levels through plasmid expression of Rv1339 using the constitutive expression system, pVV16. In Mycobacterium smegmatis mc2155, we demonstrate that recombinant expression of Rv1339 reduced cAMP levels threefold and resulted in altered gene expression, impaired bioenergetics, and a disruption in peptidoglycan biosynthesis leading to decreased tolerance to antimicrobials that target cell wall synthesis such as ethambutol, D-cycloserine, and vancomycin. This work increases our understanding of the role of cAMP in mycobacterial antimicrobial tolerance, and our observations suggest that nucleotide signaling may represent a new target for the development of antimicrobial therapies.

Echinocandins - structure, mechanism of action and use in antifungal therapy.

With increasing number of immunocompromised patients as well as drug resistance in fungi, the risk of fatal fungal infections in humans increases as well. The action of echinocandins is based on the inhibition of ß-(1,3)-d-glucan synthesis that builds the fungal cell wall. Caspofungin, micafungin, anidulafungin and rezafungin are semi-synthetic cyclic lipopeptides. Their specific chemical structure possess a potential to obtain novel derivatives with better pharmacological properties resulting in more effective treatment, especially in infections caused by Candida and Aspergillus species. In this review we summarise information about echinocandins with closer look on their chemical structure, mechanism of action, drug resistance and usage in clinical practice. We also introduce actual trends in modification of this antifungals as well as new methods of their administration, and additional use in viral and bacterial infections.

Primary Mode of Action of the Novel Sulfonamide Fungicide against Botrytis cinerea and Field Control Effect on Tomato Gray Mold.

Botrytis cinerea is considered an important plant pathogen and is responsible for significant crop yield losses. With the frequent application of commercial fungicides, B. cinerea has developed resistance to many frequently used fungicides. Therefore, it is necessary to develop new kinds of fungicides with high activity and new modes of action to solve the increasingly serious problem of resistance. During our screening of fungicide candidates, one novel sulfonamide compound, N-(2-trifluoromethyl-4-chlorphenyl)-2-oxocyclohexyl sulfonamide (L13), has been found to exhibit good fungicidal activity against B. cinerea. In this work, the mode of action of L13 against B. cinerea and the field control effect on tomato gray mold was studied. L13 had good control against B. cinerea resistant to carbendazim, diethofencarb, and iprodione commercial fungicides in the pot culture experiments. SEM and TEM observations revealed that L13 could cause obvious morphological and cytological changes to B. cinerea, including excessive branching, irregular ramification or abnormal configuration, and the decomposition of cell wall and vacuole. L13 induced more significant electrolyte leakage from hyphae than procymidone as a positive control. L13 had only a minor effect on the oxygen consumption of intact mycelia, with 2.15% inhibition at 50 µg/mL. In two locations over 2 years, the field control effect of L13 against tomato gray mold reached 83% at a rate of 450 g ai ha-1, better than the commercial fungicide of iprodione. Moreover, toxicological tests demonstrated the low toxicological effect of L13. This research seeks to provide technical support and theoretical guidance for L13 to become a real commercial fungicide.

Effect of Cinnamaldehyde on C. albicans cell wall and (1,3)- ß - D-glucans in vivo.

BACKGROUND: The incidence rate of invasive candidiasis is high, its treatment is difficult, and the prognosis is poor. In this study, an immunosuppressive mouse model of invasive Candida albicans (C. albicans) infection was constructed to observe the effects of cinnamaldehyde (CA) on the C. albicans cell wall structure and cell wall (1,3)-ß-D-glucan contents. This study provides a theoretical basis for CA treatment to target invasive C. albicans infection. METHODS: Immunosuppressed mice with invasive C. albicans infection were given an oral dosage of CA (240 mg.kg- 1.d- 1) for 14 days. Then, mouse lung tissue samples were collected for detection of the levels of (1,3)-ß-D-glucan and transmission electron microscopy observations, using fluconazole as a positive control and 2% Tween 80 saline as a negative control. RESULTS: The immunosuppressive mouse model of invasive C. albicans infection was successfully established. The levels of (1,3)-ß-D-glucan in the CA treatment group, fluconazole positive control group, invasive C. albicans infection immunosuppressive mouse model group, and 2% Tween 80 normal saline control group were 86.55 ± 126.73 pg/ml, 1985.13 ± 203.56 pg/ml, 5930.57 ± 398.67 pg/ml and 83.36 ± 26.35 pg/ml, respectively. Statistically, the CA treatment group, fluconazole positive control group and invasive C. albicans infection immunosuppressive mouse model group were compared with each other (P < 0.01) and compared with the 2% Tween 80 saline group (P < 0.01), showing that the differences were very significant. Comparison of the CA treatment group with the fluconazole positive control group (P < 0.05) displayed a difference as well. Electron microscopy showed that CA destroyed the cell wall of C. albicans, where the outer layer of the cell wall fell off and became thinner and the nuclei and organelles dissolved, but the cell membrane remained clear and intact. CONCLUSION: CA destroys the cell wall structure of C. albicans by interfering with the synthesis of (1,3)-ß-D-glucan to kill C. albicans. However, CA does not affect the cell membrane. This study provides a theoretical basis for CA treatment to target invasive C. albicans infection.

Aspergillus nidulans Septa Are Indispensable for Surviving Cell Wall Stress.

Septation in filamentous fungi is a normal part of development, which involves the formation of cross-hyphal bulkheads, typically containing pores, allowing cytoplasmic streaming between compartments. Based on previous findings regarding septa and cell wall stress, we hypothesized that septa are critical for survival during cell wall stress. To test this hypothesis, we used known Aspergillus nidulans septation-deficient mutants (ΔsepH, Δbud3, Δbud4, and Δrho4) and six antifungal compounds. Three of these compounds (micafungin, Congo red, and calcofluor white) are known cell wall stressors which activate the cell wall integrity signaling pathway (CWIS), while the three others (cycloheximide, miconazole, and 2,3-butanedione monoxime) perturb specific cellular processes not explicitly related to the cell wall. Our results show that deficiencies in septation lead to fungi which are more susceptible to cell wall-perturbing compounds but are no more susceptible to other antifungal compounds than a control. This implies that septa play a critical role in surviving cell wall stress. IMPORTANCE The ability to compartmentalize potentially lethal damage via septation appears to provide filamentous fungi with a facile means to tolerate diverse forms of stress. However, it remains unknown whether this mechanism is deployed in response to all forms of stress or is limited to specific perturbations. Our results support the latter possibility by showing that presence of septa promotes survival in response to cell wall damage but plays no apparent role in coping with other unrelated forms of stress. Given that cell wall damage is a primary effect caused by exposure to the echinocandin class of antifungal agents, our results emphasize the important role that septa might play in enabling resistance to these drugs. Accordingly, the inhibition of septum formation could conceivably represent an attractive approach to potentiating the effects of echinocandins and mitigating resistance in human fungal pathogens.

Hydrolytic Enzymes as Potentiators of Antimicrobials against an Inter-Kingdom Biofilm Model.

Biofilms are recalcitrant to antimicrobials, partly due to the barrier effect of their matrix. The use of hydrolytic enzymes capable to degrade matrix constituents has been proposed as an alternative strategy against biofilm-related infections. This study aimed to determine whether hydrolytic enzymes could potentiate the activity of antimicrobials against hard-to-treat interkingdom biofilms comprising two bacteria and one fungus. We studied the activity of a series of enzymes alone or in combination, followed or not by antimicrobial treatment, against single-, dual- or three-species biofilms of Staphylococcus aureus, Escherichia coli, and Candida albicans, by measuring their residual biomass or culturable cells. Two hydrolytic enzymes, subtilisin A and lyticase, were identified as the most effective to reduce the biomass of C. albicans biofilm. When targeting interkingdom biofilms, subtilisin A alone was the most effective enzyme to reduce biomass of all biofilms, followed by lyticase combined with an enzymatic cocktail composed of cellulase, denarase, and dispersin B that proved previously active against bacterial biofilms. The subsequent incubation with antimicrobials further reduced the biomass. Enzymes alone did not reduce culturable cells in most cases and did not interfere with the cidal effects of antimicrobials. Therefore, this work highlights the potential interest of pre-exposing interkingdom biofilms to hydrolytic enzymes to reduce their biomass besides the number of culturable cells, which was not achieved when using antimicrobials alone. IMPORTANCE Biofilms are recalcitrant to antimicrobial treatments. This problem is even more critical when dealing with polymicrobial, interkingdom biofilms, including both bacteria and fungi, as these microorganisms cooperate to strengthen the biofilm and produce a complex matrix. Here, we demonstrate that the protease subtilisin A used alone, or a cocktail containing lyticase, cellulase, denarase, and dispersin B markedly reduce the biomass of interkingdom biofilms and cooperate with antimicrobials to act upon these recalcitrant forms of infection. This work may open perspectives for the development of novel adjuvant therapies against biofilm-related infections.

An antimicrobial peptide specifically active against Listeria monocytogenes is secreted by Bacillus pumilus SF214.

BACKGROUND: Members of the Bacillus genus produce a large variety of antimicrobial peptides including linear or cyclic lipopeptides and thiopeptides, that often have a broad spectrum of action against Gram-positive and Gram-negative bacteria. We have recently reported that SF214, a marine isolated strain of Bacillus pumilus, produces two different antimicrobials specifically active against either Staphylococcus aureus or Listeria monocytogenes. The anti-Staphylococcus molecule has been previously characterized as a pumilacidin, a nonribosomally synthesized lipopetide composed of a mixture of cyclic heptapeptides linked to fatty acids of variable length. RESULTS: Our analysis on the anti-Listeria molecule of B. pumilus SF214 indicated that it is a peptide slightly smaller than 10 kDa, produced during the exponential phase of growth, stable at a wide range of pH conditions and resistant to various chemical treatments. The peptide showed a lytic activity against growing but not resting cells of Listeria monocytogenes and appeared extremely specific being inactive also against L. innocua, a close relative of L. monocytogenes. CONCLUSIONS: These findings indicate that the B. pumilus peptide is unusual with respect to other antimicrobials both for its time of synthesis and secretion and for its strict specificity against L. monocytogenes. Such specificity, together with its stability, propose this new antimicrobial as a tool for potential biotechnological applications in the fight against the dangerous food-borne pathogen L. monocytogenes.

Jerveratrum-Type Steroidal Alkaloids Inhibit ß-1,6-Glucan Biosynthesis in Fungal Cell Walls.

The limited number of available effective agents necessitates the development of new antifungals. We report that jervine, a jerveratrum-type steroidal alkaloid isolated from Veratrum californicum, has antifungal activity. Phenotypic comparisons of cell wall mutants, K1 killer toxin susceptibility testing, and quantification of cell wall components revealed that ß-1,6-glucan biosynthesis was significantly inhibited by jervine. Temperature-sensitive mutants defective in essential genes involved in ß-1,6-glucan biosynthesis, including BIG1, KEG1, KRE5, KRE9, and ROT1, were hypersensitive to jervine. In contrast, point mutations in KRE6 or its paralog SKN1 produced jervine resistance, suggesting that jervine targets Kre6 and Skn1. Jervine exhibited broad-spectrum antifungal activity and was effective against human-pathogenic fungi, including Candida parapsilosis and Candida krusei. It was also effective against phytopathogenic fungi, including Botrytis cinerea and Puccinia recondita. Jervine exerted a synergistic effect with fluconazole. Therefore, jervine, a jerveratrum-type steroidal alkaloid used in pharmaceutical products, represents a new class of antifungals active against mycoses and plant-pathogenic fungi. IMPORTANCE Non-Candida albicans Candida species (NCAC) are on the rise as a cause of mycosis. Many antifungal drugs are less effective against NCAC, limiting the available therapeutic agents. Here, we report that jervine, a jerveratrum-type steroidal alkaloid, is effective against NCAC and phytopathogenic fungi. Jervine acts on Kre6 and Skn1, which are involved in ß-1,6-glucan biosynthesis. The skeleton of jerveratrum-type steroidal alkaloids has been well studied, and more recently, their anticancer properties have been investigated. Therefore, jerveratrum-type alkaloids could potentially be applied as treatments for fungal infections and cancer.

Mycobacterium abscessus cell wall and plasma membrane characterization by EPR spectroscopy and effects of amphotericin B, miltefosine and nerolidol.

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to characterize the components of the Mycobacterium abscessus massiliense cell envelope and their interactions with amphotericin B (AmB), miltefosine (MIL), and nerolidol (NER). Spin labels analogous to stearic acid and phosphatidylcholine (PC) were distributed on an envelope layer with fluidity comparable to other biological membranes, probably the mycobacterial cell wall, because after treatment with AmB a highly rigid spectral component was evident in the EPR spectra. Methyl stearate analogue spin labels found a much more fluid membrane and did not detect the presence of AmB, except for at very high drug concentrations. Unlike other spin-labeled PCs, the TEMPO-PC spin probe, with the nitroxide moiety attached to the choline of the PC headgroup, also did not detect the presence of AmB. On the other hand, the steroid spin labels were not distributed across the membranes of M. abscessus and, instead, were concentrated in some other location of the cell envelope. Both MIL and NER compounds at 10 µM caused increased fluidity in the cell wall and plasma membrane. Furthermore, NER was shown to have a remarkable ability to extract lipids from the mycobacterial cell wall. The EPR results suggest that the resistance of mycobacteria to the action of AmB must be related to the fact that this drug does not reach the bacterial plasma membrane.

Antibacterial pyrazoles: tackling resistant bacteria.

Bacterial resistance to antibiotics threatens our progress in healthcare, modern medicine, food production and ultimately life expectancy. Antibiotic resistance is a global concern, which spreads rapidly across borders and continents due to rapid travel of people, animals and goods. Derivatives of metabolically stable pyrazole nucleus are known for their wide range of pharmacological properties, including antibacterial activities. This review highlights recent reports of pyrazole derivatives targeting different bacterial strains focusing on the drug-resistant variants. Pyrazole derivatives target different metabolic pathways of both Gram-positive and Gram-negative bacteria.

ERF Transcription Factor OsBIERF3 Positively Contributes to Immunity against Fungal and Bacterial Diseases but Negatively Regulates Cold Tolerance in Rice.

We previously showed that overexpression of the rice ERF transcription factor gene OsBIERF3 in tobacco increased resistance against different pathogens. Here, we report the function of OsBIERF3 in rice immunity and abiotic stress tolerance. Expression of OsBIERF3 was induced by Xanthomonas oryzae pv. oryzae, hormones (e.g., salicylic acid, methyl jasmonate, 1-aminocyclopropane-1-carboxylic acid, and abscisic acid), and abiotic stress (e.g., drought, salt and cold stress). OsBIERF3 has transcriptional activation activity that depends on its C-terminal region. The OsBIERF3-overexpressing (OsBIERF3-OE) plants exhibited increased resistance while OsBIERF3-suppressed (OsBIERF3-Ri) plants displayed decreased resistance to Magnaporthe oryzae and X. oryzae pv. oryzae. A set of genes including those for PRs and MAPK kinases were up-regulated in OsBIERF3-OE plants. Cell wall biosynthetic enzyme genes were up-regulated in OsBIERF3-OE plants but down-regulated in OsBIERF3-Ri plants; accordingly, cell walls became thicker in OsBIERF3-OE plants but thinner in OsBIERF3-Ri plants than WT plants. The OsBIERF3-OE plants attenuated while OsBIERF3-Ri plants enhanced cold tolerance, accompanied by altered expression of cold-responsive genes and proline accumulation. Exogenous abscisic acid and 1-aminocyclopropane-1-carboxylic acid, a precursor of ethylene biosynthesis, restored the attenuated cold tolerance in OsBIERF3-OE plants while exogenous AgNO3, an inhibitor of ethylene action, significantly suppressed the enhanced cold tolerance in OsBIERF3-Ri plants. These data demonstrate that OsBIERF3 positively contributes to immunity against M. oryzae and X. oryzae pv. oryzae but negatively regulates cold stress tolerance in rice.

Deciphering Molecular Determinants Underlying Penicillium digitatum's Response to Biological and Chemical Antifungal Agents by Tandem Mass Tag (TMT)-Based High-Resolution LC-MS/MS.

Penicillium digitatum is a widespread pathogen responsible for the postharvest decay of citrus, one of the most economically important crops worldwide. Currently, chemical fungicides are still the main strategy to control the green mould disease caused by the fungus. However, the increasing selection and proliferation of fungicide-resistant strains require more efforts to explore new alternatives acting via new or unexplored mechanisms for postharvest disease management. To date, several non-chemical compounds have been investigated for the control of fungal pathogens. In this scenario, understanding the molecular determinants underlying P. digitatum's response to biological and chemical antifungals may help in the development of safer and more effective non-chemical control methods. In this work, a proteomic approach based on isobaric labelling and a nanoLC tandem mass spectrometry approach was used to investigate molecular changes associated with P. digitatum's response to treatments with α-sarcin and beetin 27 (BE27), two proteins endowed with antifungal activity. The outcomes of treatments with these biological agents were then compared with those triggered by the commonly used chemical fungicide thiabendazole (TBZ). Our results showed that differentially expressed proteins mainly include cell wall-degrading enzymes, proteins involved in stress response, antioxidant and detoxification mechanisms and metabolic processes such as thiamine biosynthesis. Interestingly, specific modulations in response to protein toxins treatments were observed for a subset of proteins. Deciphering the inhibitory mechanisms of biofungicides and chemical compounds, together with understanding their effects on the fungal physiology, will provide a new direction for improving the efficacy of novel antifungal formulations and developing new control strategies.

Synergism of imipenem with fosfomycin associated with the active cell wall recycling and heteroresistance in Acinetobacter calcoaceticus-baumannii complex.

The carbapenem-resistant Acinetobacter calcoaceticus-baumannii (ACB) complex has become an urgent threat worldwide. Here, we determined antibiotic combinations and the feasible synergistic mechanisms against three couples of ACB (A. baumannii (AB250 and A10), A. pittii (AP1 and AP23), and A. nosocomialis (AN4 and AN12)). Imipenem with fosfomycin, the most effective in the time-killing assay, exhibited synergism to all strains except AB250. MurA, a fosfomycin target encoding the first enzyme in the de novo cell wall synthesis, was observed with the wild-type form in all isolates. Fosfomycin did not upregulate murA, indicating the MurA-independent pathway (cell wall recycling) presenting in all strains. Fosfomycin more upregulated the recycling route in synergistic strain (A10) than non-synergistic strain (AB250). Imipenem in the combination dramatically downregulated the recycling route in A10 but not in AB250, demonstrating the additional effect of imipenem on the recycling route, possibly resulting in synergism by the agitation of cell wall metabolism. Moreover, heteroresistance to imipenem was observed in only AB250. Our results indicate that unexpected activity of imipenem on the active cell wall recycling concurrently with the presence of heteroresistance subpopulation to imipenem may lead to the synergism of imipenem and fosfomycin against the ACB isolates.

Myricetin Disturbs the Cell Wall Integrity and Increases the Membrane Permeability of Candida albicans.

The fungal cell wall and membrane are the principal targets of antifungals. Herein, we report that myricetin exerts antifungal activity against Candida albicans by damaging the cell wall integrity and notably enhancing the membrane permeability. In the presence of sorbitol, an osmotic protectant, the minimum inhibitory concentration (MIC) of myricetin against C. albicans increased from 20 to 40 and 80 µg/ml in 24 and 72 h, respectively, demonstrating that myricetin disturbs the cell wall integrity of C. albicans. Fluorescence microscopic images showed the presence of propidium iodidestained C. albicans cells, indicating the myricetin-induced initial damage of the cell membrane. The effects of myricetin on the membrane permeability of C. albicans cells were assessed using crystal violet-uptake and intracellular material-leakage assays. The percentage uptakes of crystal violet for myricetin-treated C. albicans cells at 1×, 2×, and 4× the MIC of myricetin were 36.5, 60.6, and 79.4%, respectively, while those for DMSO-treated C. albicans cells were 28.2, 28.9, and 29.7%, respectively. Additionally, myricetin-treated C. albicans cells showed notable DNA and protein leakage, compared with the DMSO-treated controls. Furthermore, treatment of C. albicans cells with 1× the MIC of myricetin showed a 17.2 and 28.0% reduction in the binding of the lipophilic probes diphenylhexatriene and Nile red, respectively, indicating that myricetin alters the lipid components or order in the C. albicans cell membrane, leading to increased membrane permeability. Therefore, these data will provide insights into the pharmacological worth of myricetin as a prospective antifungal for treating C. albicans infections.

Our pursuit for effective antifungal agents targeting fungal cell wall components: where are we?

Invasive mycotic infections present unacceptably high mortality rates in humans. These infections are initiated by the fungal cell wall, which mediates host-fungi interactions. The cell wall is associated with the physiology of fungi and is involved in essential processes in the entire cell functionality. Components of the fungal cell wall are synthesised and modified in the cell wall space by the activities of cell wall proteins through a range of signalling pathways that have been described uniquely in many fungi, therefore making them suitable drug targets. The echinocandin class of cell-wall-active drugs blocks cell wall ß-1,3-glucan biosynthesis through inhibiting the catalytic subunit of the synthetic protein complex. Resistance to echinocandins can occur through the acquisition of single nucleotide polymorphisms and/or through activation of cell wall signalling pathways resulting in an altered cell wall proteome and elevated chitin content in the cell wall. Countering the cell wall remodelling process will enhance the effectiveness of ß-1,3-glucan-active antifungal agents. Cell surface proteins are also important antifungal targets that can be used to develop rapid and robust diagnostics and more effective therapeutics. The cell wall remains a crucial target in fungi that needs to be harnessed to combat mycotic infections.