Cell Membrane Stress Research Articles

The small GTPases FoRab5, FoRab7, and FoRab8 regulate vesicle transport to modulate vegetative development and pathogenicity in Fusarium oxysporum f. sp. conglutinans

Rab GTPases play a crucial role in facilitating the transportation of vesicles during the process of fungal biogenesis. Currently, there is limited understanding regarding the specific biological functions of Rab small GTPase elements within Fusarium oxysporum. In this study, we examined the three proteins FoRab5, FoRab7, and FoRab8 of Foc, which exhibit homology to the Rab protein family found in Saccharomyces cerevisiae. In addition, we also employed a PEG-mediated homologous recombination approach to create deletion mutants and complementary strains for the FoRab5, FoRab7, and FoRab8 genes, thereby facilitating a comprehensive investigation into the functional roles of these genes. FoRab5 was localized on vesicles of various sizes within the cell. Compared to the wild-type strain, the growth rate of the mutant ΔFoRab5 strain decreased, the aerial hyphae decreased, the sporulation decreased, and the spore germination rate decreased. The sensitivity to cell membrane stress, cell wall stress, and endoplasmic reticulum stress increased, the activity of laccase and glucoamylase decreased significantly, and the pathogenicity to cabbage seedlings decreased. FoRab7 was localized on the vacuolar membrane. Compared to the wild type, the growth rate of the mutant ΔFoRab7 strain decreased, the bacteria produced a large amount of pigment deposition, the aerial hyphae decreased significantly, the hyphal branches increased, and the mutant almost lost the ability to produce spores. The sensitivity to osmotic stress, cell membrane stress, cell wall stress, metal ion stress, and endoplasmic reticulum stress was enhanced, and the vacuole was fragmented. Laccase and glucoamylase activities decreased in a significant manner. Moreover, there was a decrease in the pathogenicity of cabbage seedlings. FoRab8 was localized at the tip of the mycelium. Compared to the wild type, the growth rate of the mutant ΔFoRab8 strain decreased, the sporulation decreased, and the sensitivity of the mutant to osmotic stress and endoplasmic reticulum pressure increased. There was a significant decrease in the activity of laccase, glucoamylase, and cellulase. A reduction in the pathogenicity to cabbage seedlings occurred. In summary, these results indicate that members of the Rab family proteins FoRab5, FoRab7, and FoRab8 regulate a series of processes such as growth, sporulation, pathogenicity, and ectoenzyme secretion in Foc.

Xanthomonas citri subsp. citri requires a genus-specific outer membrane protein and TolB to coordinate cell membrane integrity and virulence.

Xanthomonas citri subsp. citri (Xcc) possesses a Xanthomonas-specific outer membrane protein XAC1347 (OMPXan) that exerts a role in the expression of the type III secretion system for pathogenicity. In this study, we reported that OMPXan was required for salt stress tolerance and cell membrane integrity, as well as the expression of the gum genes for the production of extracellular polysaccharides. Pull-down and yeast two-hybrid assays revealed that OMPXan interacts with TolB, a substrate of the Tol/Pal membrane protein system. The deletion of tolB resulted in similar phenotypic alterations as the OMPXan mutant in salt stress tolerance, cell membrane integrity, and the expression of hrpG, hrpX, hrpD6, and hrcC for pathogenicity. In contrast, the absence of TolB resulted in an increased level of expression of the gum genes and the production of extracellular polysaccharides. These results indicate that the interaction of OMPXan and TolB coordinates multi-faceted mechanisms to manage environmental stress and pathogenicity.IMPORTANCEThe gram-negative Xanthomoas citri subsp. citri (Xcc) is a causal agent of citrus canker, a serious bacterial disease on citrus plants. Our previous research reported that a Xanthomonas-specific outer membrane protein XAC1347 (OMPXan) is necessary for type III gene expression. This manuscript provided evidence to show that OMPXan interacts with Tol/Pal system substrate TolB. Moreover, OMPXan and TolB are both required for cell membrane integrity, stress adaption, and virulence. The overall results support that OMPXan and TolB coordinate multi-faceted mechanisms to manage environmental stress and pathogenicity.

Dominant spoilage bacteria in crayfish alleviate ultrasonic stress through mechanosensitive channels but could not prevent the process of membrane destruction

Although there have been many studies on the efficacy of ultrasonic inactivation, the stress resistance mechanism of bacteria is still a challenge for complete ultrasonic inactivation. In this study, the dominant spoilage bacteria in crayfish, Shewanella baltica (S. baltica) and Aeromonas veronii (A. veronii), were subjected to high-intensity ultrasonic treatment. The results showed compromised cell membrane, decreased membrane fluidity, hyperpolarized membrane potential, and disrupted succinate-coenzyme Q reductase. Transmission electron microscopy revealed significant fragmentation of S. baltica, whereas A. veronii, with its thick cell wall and outer capsule membrane, demonstrated enhanced resistance to ultrasound. Real-time quantitative PCR indicated that in response to ultrasonic stress, bacteria initiated a stress response mechanism by increasing the expression of mechanosensitive channels; meanwhile, the outer capsule of A. veronii delayed the transformation of ultrasonic external forces into cell membrane stress. The study found that in response to ultrasonic stress, bacteria initiated a stress response mechanism by increasing the expression of mechanosensitive channels as “emergency valve” in short time but could not prevent the process of membrane destruction with prolonged exposure. This finding provided a basis for addressing bacterial stress tolerance in ultrasonic inactivation.

PIEZO1 Affects Cell Growth and Migration via Microfilament-Mediated YAP trans-Latitudinal Regulation.

Environmental mechanical forces, such as cell membrane stress, cell extrusion, and stretch, have been proven to affect cell growth and migration. Piezo1, a mechanosensitive channel protein, responds directly to endogenous or exogenous mechanical stimuli. Here, we explored the Piezo1 distribution and microfilament morphological changes induced by mechanical forces in the tumor and normal cells. In addition, Piezo1 activation in tumor cells resulted in the nuclear accumulation of YAP, whereas nuclear export of YAP and microfilament depolymerization occurred with the prolonged activation, while a removal stimulation restored the YAP distribution and microfilament polymerization. Combining the morphological changes of the microfilament under Piezo1 activation and the function of YAP in regulating cell growth and development, we suggest that Piezo1 senses changes in environmental mechanical forces and regulates YAP distribution through the microfilament cytoskeleton network, which in turn affects the growth and migration more obviously in tumor cells rather than normal cells. Our results are essential for understanding the trans-latitudinal transmission of mechanical forces and exploring the role of environmental mechanical forces in tumor therapy.

A key component Rxt3 in the Rpd3L histone deacetylase complex regulates development, stress tolerance, amylase production and kojic acid synthesis in Aspergillus oryzae.

Rpd3L is a highly conserved histone deacetylase complex in eukaryotic cells and participates in various cellular processes. However, the roles of the Rpd3L component in filamentous fungi remain to be delineated ultimately. In this study, we constructed two knockout mutants of Rpd3L's Rxt3 subunit and characterized their biological functions in A. oryzae. Phenotypic analysis showed that AoRxt3 played a positive role in hyphal growth and conidia formation. Deletion of Aorxt3 resulted in augmented tolerance to multiple stresses, including cell wall stress, cell membrane stress, endoplasmic reticulum stress, osmotic stress and oxidative stress. Noteworthily, we found that Aorxt3-deleting mutants showed a higher kojic acid production than the control strain. However, the loss of Aorxt3 led to a significant decrease in amylase synthesis. Our findings lay the foundation for further exploring the role of other Rpd3L subunits and provide a new strategy to improve kojic acid production in A. oryzae.

Erg251 has complex and pleiotropic effects on sterol composition, azole susceptibility, filamentation, and stress response phenotypes.

Ergosterol is essential for fungal cell membrane integrity and growth, and numerous antifungal drugs target ergosterol. Inactivation or modification of ergosterol biosynthetic genes can lead to changes in antifungal drug susceptibility, filamentation and stress response. Here, we found that the ergosterol biosynthesis gene ERG251 is a hotspot for point mutations during adaptation to antifungal drug stress within two distinct genetic backgrounds of Candida albicans. Heterozygous point mutations led to single allele dysfunction of ERG251 and resulted in azole tolerance in both genetic backgrounds. This is the first known example of point mutations causing azole tolerance in C. albicans. Importantly, single allele dysfunction of ERG251 in combination with recurrent chromosome aneuploidies resulted in bona fide azole resistance. Homozygous deletions of ERG251 caused increased fitness in low concentrations of fluconazole and decreased fitness in rich medium, especially at low initial cell density. Homozygous deletions of ERG251 resulted in accumulation of ergosterol intermediates consistent with the fitness defect in rich medium. Dysfunction of ERG251, together with FLC exposure, resulted in decreased accumulation of the toxic sterol (14-ɑ-methylergosta-8,24(28)-dien-3β,6α-diol) and increased accumulation of non-toxic alternative sterols. The altered sterol composition of the ERG251 mutants had pleiotropic effects on transcription, filamentation, and stress responses including cell membrane, osmotic and oxidative stress. Interestingly, while dysfunction of ERG251 resulted in azole tolerance, it also led to transcriptional upregulation of ZRT2, a membrane-bound Zinc transporter, in the presence of FLC, and overexpression of ZRT2 is sufficient to increase azole tolerance in wild-type C. albicans. Finally, in a murine model of systemic infection, homozygous deletion of ERG251 resulted in decreased virulence while the heterozygous deletion mutants maintain their pathogenicity. Overall, this study demonstrates that single allele dysfunction of ERG251 is a recurrent and effective mechanism of acquired azole tolerance. We propose that altered sterol composition resulting from ERG251 dysfunction mediates azole tolerance as well as pleiotropic effects on stress response, filamentation and virulence.

The target of rapamycin signaling pathway regulates vegetative development, aflatoxin biosynthesis, and pathogenicity in Aspergillus flavus.

The target of rapamycin (TOR) signaling pathway is highly conserved and plays a crucial role in diverse biological processes in eukaryotes. Despite its significance, the underlying mechanism of the TOR pathway in Aspergillus flavus remains elusive. In this study, we comprehensively analyzed the TOR signaling pathway in A. flavus by identifying and characterizing nine genes that encode distinct components of this pathway. The FK506-binding protein Fkbp3 and its lysine succinylation are important for aflatoxin production and rapamycin resistance. The TorA kinase plays a pivotal role in the regulation of growth, spore production, aflatoxin biosynthesis, and responses to rapamycin and cell membrane stress. As a significant downstream effector molecule of the TorA kinase, the Sch9 kinase regulates aflatoxin B1 (AFB1) synthesis, osmotic and calcium stress response in A. flavus, and this regulation is mediated through its S_TKc, S_TK_X domains, and the ATP-binding site at K340. We also showed that the Sch9 kinase may have a regulatory impact on the high osmolarity glycerol (HOG) signaling pathway. TapA and TipA, the other downstream components of the TorA kinase, play a significant role in regulating cell wall stress response in A. flavus. Moreover, the members of the TapA-phosphatase complexes, SitA and Ppg1, are important for various biological processes in A. flavus, including vegetative growth, sclerotia formation, AFB1 biosynthesis, and pathogenicity. We also demonstrated that SitA and Ppg1 are involved in regulating lipid droplets (LDs) biogenesis and cell wall integrity (CWI) signaling pathways. In addition, another phosphatase complex, Nem1/Spo7, plays critical roles in hyphal development, conidiation, aflatoxin production, and LDs biogenesis. Collectively, our study has provided important insight into the regulatory network of the TOR signaling pathway and has elucidated the underlying molecular mechanisms of aflatoxin biosynthesis in A. flavus.

Functional analysis of basidiomycete specific chitin synthase genes in the agaricomycete fungus Pleurotus ostreatus

Chitin is an essential structural component of fungal cell walls composed of transmembrane proteins called chitin synthases (CHSs), which have a large range of reported effects in ascomycetes; however, are poorly understood in agaricomycetes. In this study, evolutionary and molecular genetic analyses of chs genes were conducted using genomic information from nine ascomycete and six basidiomycete species. The results support the existence of seven previously classified chs clades and the discovery of three novel basidiomycete-specific clades (BI–BIII). The agaricomycete fungus Pleurotus ostreatus was observed to have nine putative chs genes, four of which were basidiomycete-specific. Three of these basidiomycete specific genes were disrupted in the P. ostreatus 20b strain (ku80 disruptant) through homologous recombination and transformants were obtained (Δchsb2, Δchsb3, and Δchsb4). Despite numerous transformations Δchsb1 was unobtainable, suggesting disruption of this gene causes a crucial negative effect in P. ostreatus. Disruption of these chsb2–4 genes caused sparser mycelia with rougher surfaces and shorter aerial hyphae. They also caused increased sensitivity to cell wall and membrane stress, thinner cell walls, and overexpression of other chitin and glucan synthases. These genes have distinct roles in the structural formation of aerial hyphae and cell walls, which are important for understanding basidiomycete evolution in filamentous fungi.

Comparative transcriptome analysis reveals the redirection of metabolic flux from cell growth to astaxanthin biosynthesis in Yarrowia lipolytica.

Engineering Yarrowia lipolytica to produce astaxanthin provides a promising route. Here, Y. lipolytica M2 producing a titer of 181 mg/L astaxanthin was isolated by iterative atmospheric and room-temperature plasma mutagenesis and diphenylamine-mediated screening. Interestingly, a negative correlation was observed between cell biomass and astaxanthin production. To reveal the underlying mechanism, RNA-seq analysis of transcriptional changes was performed in high producer M2 and reference strain M1, and a total of 1379 differentially expressed genes were obtained. Data analysis revealed that carbon flux was elevated through lipid metabolism, acetyl-CoA and mevalonate supply, but restrained through central carbon metabolism in strain M2. Moreover, upregulation of other pathways such as ATP-binding cassette transporter and thiamine pyrophosphate possibly provided more cofactors for carotenoid hydroxylase and relieved cell membrane stress caused by astaxanthin insertion. These results suggest that balancing cell growth and astaxanthin production may be important to promote efficient biosynthesis of astaxanthin in Y. lipolytica.

LiaX is a surrogate marker for cell envelope stress and daptomycin non-susceptibility in Enterococcus faecium.

Daptomycin (DAP) is often used as a first-line therapy to treat vancomycin-resistant Enterococcus faecium infections, but emergence of DAP non-susceptibility threatens the effectiveness of this antibiotic. Moreover, current methods to determine DAP minimum inhibitory concentrations (MICs) have poor reproducibility and accuracy. In enterococci, DAP resistance is mediated by the LiaFSR cell membrane stress response system, and deletion of liaR encoding the response regulator results in hypersusceptibility to DAP and antimicrobial peptides. The main genes regulated by LiaR are a cluster of three genes, designated liaXYZ. In Enterococcus faecalis, LiaX is surface-exposed with a C-terminus that functions as a negative regulator of cell membrane remodeling and an N-terminal domain that is released to the extracellular medium where it binds DAP. Thus, in E. faecalis, LiaX functions as a sentinel molecule recognizing DAP and controlling the cell membrane response, but less is known about LiaX in E. faecium. Here, we found that liaX is essential in E. faecium with an activated LiaFSR system. Unlike E. faecalis, E. faecium LiaX is not detected in the extracellular milieu and does not appear to alter phospholipid architecture. We further postulated that LiaX could be used as a surrogate marker for cell envelope activation and non-susceptibility to DAP. For this purpose, we developed and optimized a LiaX enzyme-linked immunosorbent assay (ELISA). We then assessed 86 clinical E. faecium bloodstream isolates for DAP MICs and used whole genome sequencing to assess for substitutions in LiaX. All DAP-resistant clinical strains of E. faecium exhibited elevated LiaX levels. Strikingly, 73% of DAP-susceptible isolates by standard MIC determination also had elevated LiaX ELISAs compared to a well-characterized DAP-susceptible strain. Phylogenetic analyses of predicted amino acid substitutions showed 12 different variants of LiaX without a specific association with DAP MIC or LiaX ELISA values. Our findings also suggest that many E. faecium isolates that test DAP susceptible by standard MIC determination are likely to have an activated cell stress response that may predispose to DAP failure. As LiaX appears to be essential for the cell envelope response to DAP, its detection could prove useful to improve the accuracy of susceptibility testing by anticipating therapeutic failure.

Exploring the Biocontrol Capability of Non-Mycotoxigenic Strains of Penicillium expansum.

Penicillium expansum is one the major postharvest pathogens of pome fruit during postharvest handling and storage. This fungus also produces patulin, which is a highly toxic mycotoxin that can contaminate infected fruits and their derived products and whose levels are regulated in many countries. In this study, we investigated the biocontrol potential of non-mycotoxigenic strains of Penicillium expansum against a mycotoxigenic strain. We analyzed the competitive behavior of two knockout mutants that were unable to produce patulin. The first mutant (∆patK) involved the deletion of the patK gene, which is the initial gene in patulin biosynthesis. The second mutant (∆veA) involved the deletion of veA, which is a global regulator of primary and secondary metabolism. At the phenotypic level, the ∆patK mutant exhibited similar phenotypic characteristics to the wild-type strain. In contrast, the ∆veA mutant displayed altered growth characteristics compared with the wild type, including reduced conidiation and abnormal conidiophores. Neither mutant produced patulin under the tested conditions. Under various stress conditions, the ∆veA mutants exhibited reduced growth and conidiation when exposed to stressors, including cell membrane stress, oxidative stress, osmotic stress, and different pH values. However, no significant changes were observed in the ∆patK mutant. In competitive growth experiments, the presence of non-mycotoxigenic strains reduced the population of the wild-type strain during in vitro growth. Furthermore, the addition of either of the non-mycotoxigenic strains resulted in a significant decrease in patulin levels. Overall, our results suggest the potential use of non-mycotoxigenic mutants, particularly ∆patK mutants, as biocontrol agents to reduce patulin contamination in food and feed.

Biogenic Synthesis of Photosensitive Magnesium Oxide Nanoparticles Using Citron Waste Peel Extract and Evaluation of Their Antibacterial and Anticarcinogenic Potential.

Magnesium oxide nanoparticles (MgONPs) have been fabricated by several approaches, including green chemistry approach due to diverse application and versatile features. The current study aimed to prepare a convenient, biocompatible, and economically viable MgONPs using waste citron peel extract (CP-MgONPs) to evaluate their biological applications. The CP-MgONPs were synthesized by a sustainable approach from extract of waste citron peel both as capping and reducing agents without use of any hazardous material. The physicochemical features of formed CP-MgONPs were determined by sophisticated analytical and microscopic techniques. The biogenic CP-MgONPs were examined for their antibacterial, anticarcinogenic, and photocatalytic attributes. A prominent absorption peak in the UV-Vis spectra at 284 nm was the distinguishing characteristic of the CP-MgONPs. The scanning electron microscopy (SEM) reveals polyhedral morphology of nanoparticles with slight agglomeration of CP-MgONPs. The CP-MgONPs exerted excellent antibacterial potencies against six bacterial strains. The CP-MgONPs displayed significant susceptibility towards E. coli (20.72 ± 0.33 mm) and S. aureus (19.52 ± 0.05 mm) with the highest inhibition zones. The anticancer effect of CP-MgONPs was evaluated against HepG2 (IC50 : 15.3 μg·mL-1) cancer cells and exhibited potential anticancer activity. A prompt inversion of cellular injury manifested as impairment of the integrity of the cell membrane, apoptosis, and oxidative stress was observed in treated cells with CP-MgONPs. The biosynthesized CP-MgONPs also conducted successful photocatalytic potential as much as MgO powder under the UV-light using acid orange 8 (AO-8) dye. The degradation performance of CP-MgONPs showed over 94% photocatalytic degradation efficiency of acid orange 8 (AO-8) dyes within a short time. Outcomes of this research signify that biogenic CP-MgONPs may be advantageous at low concentrations, with positive environmental impacts.

Activation of Ustilaginoidin Biosynthesis Gene uvpks1 in Villosiclava virens Albino Strain LN02 Influences Development, Stress Responses, and Inhibition of Rice Seed Germination.

Villosiclava virens (anamorph: Ustilaginoidea virens) is the pathogen of rice false smut (RFS), which is a destructive rice fungal disease. The albino strain LN02 is a natural white-phenotype mutant of V. virens due to its incapability to produce toxic ustilaginoidins. In this study, three strains including the normal strain P1, albino strain LN02, and complemented strain uvpks1C-1 of the LN02 strain were employed to investigate the activation of the ustilaginoidin biosynthesis gene uvpks1 in the albino strain LN02 to influence sporulation, conidia germination, pigment production, stress responses, and the inhibition of rice seed germination. The activation of the ustilaginoidin biosynthesis gene uvpks1 increased fungal tolerances to NaCl-induced osmotic stress, Congo-red-induced cell wall stress, SDS-induced cell membrane stress, and H2O2-induced oxidative stress. The activation of uvpks1 also increased sporulation, conidia germination, pigment production, and the inhibition of rice seed germination. In addition, the activation of uvpks1 was able to increase the mycelial growth of the V. virens albino strain LN02 at 23 °C and a pH from 5.5 to 7.5. The findings help in understanding the effects of the activation of uvpks1 in albino strain LN02 on development, pigment production, stress responses, and the inhibition of rice seed germination by controlling ustilaginoidin biosynthesis.

Identifying LiaFSR Residues Contributing to ExPortal Integrity and Response to Antimicrobials in Group A Streptococcus

Abstract Background Group A Streptococcus (GAS), or Streptococcus pyogenes, is responsible for a myriad of diseases (pharyngitis, scarlet fever, impetigo, toxic shock syndrome) of which several are prevalent in the pediatric population. The LiaFSR two-component regulatory system (TCS) and the ExPortal functional membrane microdomain (FMM) of GAS have been shown to coordinate the response to cell membrane stress when the bacterium is exposed to antimicrobials or antimicrobial peptides (AMPs). The ExPortal coordinates translocation and processing of secreted GAS proteins and contributes to sensing membrane. In an unstressed state, LiaF acts as an inhibitor of LiaS activity, both residing within the ExPortal. In a stressed state, such as exposure to antimicrobial agents, LiaF and S dissociate, allowing LiaS to phosphorylate LiaR, resulting in subsequent activation and expression of downstream target genes. Genomic analysis indicates a strict conservation of specific protein domains and certain amino acid residues in LiaF, S, and R, which suggests their critical involvement in the LiaFSR mechanism of action. We hypothesize that conserved amino acid residues in LiaFSR, such as the LiaF C-terminal domain (CTD) and a 3xK residue motif, are essential to ExPortal formation and maintenance and may contribute to differences in antimicrobial susceptibility and virulence. Methods Two different mutant strains were analyzed and compared to (wild-type) WT: one lacking the LiaF CTD (LiaF_∆CTD) and one mutagenized in the 3xK motif (LiaF_K91-93A). Five different assays were used to examine phenotypes associated with LiaFSR activity: SpeB protease activity through bacterial growth on milk plates, ExPortal integrity via fluorescence microscopy, diamide-induced oxidative stress tolerance, antimicrobial/antimicrobial peptide (AM/AMP) susceptibility, and a direct bactericidal assay in human blood (Lancefield). For the AM/AMP susceptibility, we tested 6 different antimicrobial agents with varying mechanisms of action: bacitracin, polymyxin B, LL-37, nisin, daptomycin, and HNP-1. Results Milk plate analysis revealed a significant decrease in SpeB production in the LiaF-∆CTD vs. WT, but no difference in LiaF-K91-93A relative to WT. During fluorescence microscopy, no significant differences were detected between foci of fluorescence of either mutant strain relative to WT. When exposed to diamide-induced oxidative stress, both mutants exhibited increased tolerance relative to WT. AM/AMP susceptibility assays did not reveal any significant differences for any antimicrobial agent between either mutant strain vs. WT. Finally, the Lancefield assay revealed a significant decrease in survival in human blood between both mutant strains and WT. Mutant strains also had significantly decreased survival relative to a strain lacking LiaF (∆LiaF). Conclusions The results of the AM/AMP susceptibility assay suggest that, unlike the LiaFSR system in Enterococcus, LiaF function does not contribute to LiaFSR-dependent susceptibility. Significant differences in survival in blood indicate that the LiaF residues studied are physiologically relevant in GAS infection. However, their exact mechanism underlying this contribution is being investigated. Future research will examine downstream gene regulation to elucidate the LiaFSR-associated mechanism of decreased survival in human blood, with the aim of defining determinants of GAS bacteremia.

Potential utility of endophytic Bacillus altitudinis strain P32-3 as a biocontrol agent for the postharvest prevention of sweet potato black rot

Sweet potato black rot is a common postharvest disease caused by the pathogenic fungus Ceratocystis fimbriata. The toxins produced by infected tubers are potentially harmful to humans. Plant endophytic bacteria may serve as biocontrol agents for protecting crops (including vegetables) during postharvest storage without detrimental effects on humans or other animals. In this study, 11 bacterial strains of Bacillus, Priestia, Blautia, Pseudomonas, and Sphingomonas species were isolated from sweet potato storage roots, of which only one strain (P32-3), which was identified as Bacillus altitudinis, strongly inhibited C. fimbriata mycelial growth and spore germination. Furthermore, the volatile organic compounds and cell-free supernatant of strain P32-3 repressed C. fimbriata growth. Additionally, P32-3 treatments significantly decreased the disease spot diameter and depth on harvested sweet potato roots. A total of 981 differentially expressed genes were identified by comparing the transcriptomes of the control and P32-3-treated mycelia. Strain P32-3 significantly altered the expressions of genes related to cell wall integrity, the cell membrane, spore germination, energy metabolism, pathogenicity, and oxidative stress. In addition, RNA-seq results were validated by a quantitative real-time polymerase chain reaction analysis. Our results indicate that B. altitudinis strain P32-3 can inhibit C. fimbriata growth and development and protect postharvest sweet potato storage roots from C. fimbriata infection. The study findings provide the theoretical basis for future investigations on the utility of strain P32-3 for protecting sweet potato against black rot during the postharvest stage.

A potential hyphal fusion protein complex with an important role in development and virulence interacts with autophagy related proteins in Fusarium pseudograminearum

Hyphal fusion (anastomosis) is a common process serving many important functions at various developmental stages in the life cycle of ascomycetous fungi. However, the biological roles and molecular mechanisms in plant pathogenic fungi was widely unknown. In this study, a hyphal fusion protein FpHam-2 was screened from a T-DNA insertion mutant library of Fusarium pseudograminearum, and FpHam-2 interacts with another two hyphal fusion protein homologues FpHam-3 and FpHam-4. Each of these three genes deletion mutant revealed in similar defective phenotypes compared with the WT and complemented strains, including reduction in growth rate, defects in hyphal fusion and conidiation, more sensitive for cell membrane, cell wall and oxidative stress responses, and decreased in virulence. The Yeast two-hybrid assay was used to identify that FpHam-2 interacts with three autophagy related proteins, including FpAtg3, FpAtg28 and FpAtg33. Furthermore, FpHam-2-deleted mutant showed decreased accumulation of autophagic bodies in hypha. In conclusion, FpHam-2, FpHam-3 and FpHam-4 are essential role for hyphal fusion and regulating the growth, conidiation and virulence in F. pseudograminearum.

Hog1-mediated stress tolerance in the pathogenic fungus Trichosporon asahii

Trichosporon asahii is an opportunistic pathogenic fungus that causes severe and sometimes fatal infections in immunocompromised patients. Hog1, a mitogen-activated protein kinase, regulates the stress resistance of some pathogenic fungi, however its role in T. asahii has not been investigated. Here, we demonstrated that the hog1 gene-deficient T. asahii mutant is sensitive to high temperature, cell membrane stress, oxidative stress, and antifungal drugs. Growth of the hog1 gene-deficient T. asahii mutant was delayed at 40 °C. The hog1 gene-deficient T. asahii mutant also exhibited sensitivity to sodium dodecyl sulfate, hydrogen peroxide, menadione, methyl methanesulfonate, UV exposure, and antifungal drugs such as amphotericin B under a glucose-rich condition. Under a glucose-restricted condition, the hog1 gene-deficient mutant exhibited sensitivity to NaCl and KCl. The virulence of the hog1 gene-deficient mutant against silkworms was attenuated. Moreover, the viability of the hog1 gene-deficient mutant decreased in the silkworm hemolymph. These phenotypes were restored by re-introducing the hog1 gene into the gene-deficient mutant. Our findings suggest that Hog1 plays a critical role in regulating cellular stress responses in T. asahii.

Elucidating Mechanotransduction Processes During Magnetomechanical Neuromodulation Mediated by Magnetic Nanodiscs.

Noninvasive cell-type-specific manipulation of neural signaling is critical in basic neuroscience research and in developing therapies for neurological disorders. Magnetic nanotechnologies have emerged as non-invasive neuromodulation approaches with high spatiotemporal control. We recently developed a wireless force-induced neurostimulation platform utilizing micro-sized magnetic discs (MDs) and low-intensity alternating magnetic fields (AMFs). When targeted to the cell membrane, MDs AMFs-triggered mechanoactuation enhances specific cell membrane receptors resulting in cell depolarization. Although promising, it is critical to understand the role of mechanical forces in magnetomechanical neuromodulation and their transduction to molecular signals for its optimization and future translation. MDs are fabricated using top-down lithography techniques, functionalized with polymers and antibodies, and characterized for their physical properties. Primary cortical neurons co-cultured with MDs and transmembrane protein chemical inhibitors are subjected to 20s pulses of weak AMFs (18 mT, 6Hz). Calcium cell activity is recorded during AMFs stimulation. Neuronal activity in primary rat cortical neurons is evoked by the AMFs-triggered actuation of targeted MDs. Ion channel chemical inhibition suggests that magnetomechanical neuromodulation results from MDs actuation on Piezo1 and TRPC1 mechanosensitive ion channels. The actuation mechanisms depend on MDs size, with cell membrane stretch and stress caused by the MDs torque being the most dominant. Magnetomechanical neuromodulation represents a tremendous potential since it fulfills the requirements of negligible heating (ΔT < 0.1°C) and weak AMFs (< 100Hz), which are limiting factors in the development of therapies and the design of clinical equipment. The online version contains supplementary material available at 10.1007/s12195-023-00786-8.

Tea Polyphenols Inhibit the Activity and Toxicity of Staphylococcus aureus by Destroying Cell Membranes and Accumulating Reactive Oxygen Species.

Staphylococcus aureus can cause bacterial food intoxication and seriously affect human health. Tea polyphenols (TP) are a kind of natural, safe, and broad-spectrum bacteriostatic substances, with a wide range of bacteriostatic effects. In the study, we explored the possible bacteriostatic mode of TP. The minimum inhibitory concentration of TP against S. aureus was 64 μg/mL. Protein, DNA, and K+ leak experiments, fluorescence microscopy, and transmission electron microscopy suggested that TP disrupt cell membranes, leading to intracellular component loss. By studying the effect of TP on the toxicity of S. aureus, it was found that the expression levels of two toxin genes, coa and spa, were downregulated by 2.37 and 32.6, respectively. Furthermore, after treatment with TP, a large number of reactive oxygen species (ROS) were propagated and released, leading to oxidative stress in cells. We speculated that the bacteriostatic mechanism of TP may be through the destruction of the cell membrane and ROS-mediated oxidative stress. Meanwhile, the hemolysis activity proved the safety of TP. Our results suggested that TP may be a potential antimicrobial agent for food.

Recent advances and mechanism of antimicrobial efficacy of graphene-based materials: a review.

Graphene-based materials have undergone substantial investigation in recent years owing to their wide array of physicochemical characteristics. Employment of these materials in the current state, where infectious illnesses caused by microbes have severely damaged human life, has found widespread application in combating fatal infectious diseases. These materials interact with the physicochemical characteristics of the microbial cell and alter or damage them. The current review is dedicated to molecular mechanisms underlying the antimicrobial property of graphene-based materials. Various physical and chemical mechanisms leading to cell membrane stress, mechanical wrapping, photo-thermal ablation as well as oxidative stress exerting antimicrobial effect have also been thoroughly discussed. Furthermore, an overview of the interactions of these materials with membrane lipids, proteins, and nucleic acids has been provided. A thorough understanding of discussed mechanisms and interactions is essential to develop extremely effective antimicrobial nanomaterial for application as an antimicrobial agent.