Cell Wall Inhibitors Research Articles

AaSlt2 Is Required for Vegetative Growth, Stress Adaption, Infection Structure Formation, and Virulence in Alternaria alternata

Slt2 is an important component of the Slt2-MAPK pathway and plays critical regulatory roles in growth, cell wall integrity, melanin biosynthesis, and pathogenicity of plant fungi. AaSlt2, an ortholog of the Saccharomyces cerevisiae Slt2 gene, was identified from A. alternata in this study, and its function was clarified by knockout of the gene. The ΔAaSlt2 strain of A. alternata was found to be defective in spore morphology, vegetative growth, and sporulation. Analysis of gene expression showed that expression of the AaSlt2 gene was significantly up-regulated during infection structure formation of A. alternata on hydrophobic and pear wax extract-coated surfaces. Further tests on onion epidermis confirmed that spore germination was reduced in the ΔAaSlt2 strain, together with decreased formation of appressorium and infection hyphae. Moreover, the ΔAaSlt2 strain was sensitive to cell wall inhibitors, and showed significantly reduced virulence on pear fruit. Furthermore, cell wall degradation enzyme (CWDE) activities, melanin accumulation, and toxin biosynthesis were significantly lower in the ΔAaSlt2 strain. Overall, the findings demonstrate the critical involvement of AaSlt2 in growth regulation, stress adaptation, infection structure formation, and virulence in A. alternata.

Calcium‐Dependent Lipopeptide Antibiotics against Drug‐Resistant Pathogens Discovered via Host‐Dependent Heterologous Expression of a Cloned Biosynthetic Gene Cluster

AbstractHistorically, small molecules biosynthesised by bacteria have been an excellent source for antibacterial drugs. Today, however, the rediscovery of known compounds is a significant hurdle to developing new antimicrobials. Here we use a genome mining and synthetic biology approach to discover the ambocidins: calcium‐dependent lipodepsipeptides that are active against drug‐resistant Gram‐positive pathogens. By cloning a silent biosynthetic gene cluster (the amb cluster) from Streptomyces ambofaciens ATCC 2387 and integrating this into the chromosome of Streptomyces avermitilis we induce expression of ambocidin A and B: two new Nϵ‐hydroxyarginine‐containing cyclic lipodepsipeptides active against drug‐resistant Gram‐positive pathogens. Using a panel of Streptomyces host strains, we show that the choice of heterologous host is critical for producing the biologically active compounds, and that inappropriate host choice leads to aberrant production inactive derivatives. We show that Nϵ‐hydroxyarginine is the product of a heme‐dependent oxygenase and that it enhances biological activity. Ambocidin A inhibits cell wall biosynthesis by binding to Lipid II at a different site than vancomycin. Furthermore, unlike daptomycin, ambocidin A retains potent antimicrobial activity in the presence of lung surfactant, giving it the potential to treat bacterial pneumonia. Our work expands the family of calcium‐dependent lipopeptide antibiotics with a new member exhibiting a distinct mechanism of action.

The roles of cell wall inhibition responsive protein CwrA in the pathogenicity of staphylococcus aureus

ABSTRACT The ability to form robust biofilms and secrete a diverse array of virulence factors are key pathogenic determinants of Staphylococcus aureus, causing a wide range of infectious diseases. Here, we characterized cwrA as a VraR-regulated gene encoding a cell wall inhibition-responsive protein (CwrA) using electrophoretic mobility shift assays. We constructed cwrA deletion mutants in the genetic background of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains. Phenotypic analyses indicated that deletion of cwrA led to impaired biofilm formation, which was correlated with polysaccharide intercellular adhesin (PIA). Besides, the results of real-time quantitative PCR (RT-qPCR) and β-galactosidase activity assay revealed that CwrA promoted biofilm formation by influence the ica operon activity in S. aureus. Furthermore, cwrA deletion mutants released less extracellular DNA (eDNA) in the biofilm because of their reduced autolytic activity compared to the wild-type (WT) strains. We also found that cwrA deletion mutant more virulence than the parental strain because of its enhanced haemolytic activity. Mechanistically, this phenotypic alteration is related to activation of the SaeRS two-component system, which positively regulates the transcriptional levels of genes encoding membrane-damaging toxins. Overall, our results suggest that CwrA plays an important role in modulating biofilm formation and haemolytic activity in S. aureus.

Cell wall nanoparticles from hyphae of Alternaria infectoria grown with caspofungin, nikkomycin, or pyroquilon trigger different activation profiles in macrophages.

Alternaria infectoria causes opportunistic human infections and is a source of allergens leading to respiratory allergies. In this work, we prepared cell wall nanoparticles (CWNPs) as a novel approach to study macrophage immunomodulation by fungal hyphal cell walls. A. infectoria was grown in the presence of caspofungin, an inhibitor of β(1,3)-glucan synthesis; nikkomycin Z, an inhibitor of chitin synthases; and pyroquilon, an inhibitor of dihydroxynaphthalene (DHN)-melanin synthesis. Distinct CWNPs were obtained from these cultures, referred to as casCWNPs, nkCWNPs, and pyrCWNPs, respectively. CWNPs are round-shaped particles with a diameter of 70-200 nm diameter particles that when added to macrophages are taken up by membrane ruffling. CWNPs with no DHN-melanin and more glucan (pyrCWNPs) caused early macrophage activation and lowest viability, with the cells exhibiting ultrastructural modifications such as higher vacuolization and formation of autophagy-like structures. CasCWNPs promoted the highest tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) increase, also resulting in the release of partially degraded chitin, an aspect never observed in macrophage-like cells and fungi. After 6 h of interaction with CWNPs, only half were viable, except with control CWNPs. Overall, this work indicates that compounds that modify the fungal cell wall led to CWNPs with new properties that may have implications for the effects of drugs during antifungal therapy. CWNPs provide a new tool to study the interaction of hyphal fungal cell wall components with phagocytic cells and enable to show how the modification of cell wall components in A. infectoria can modulate the response by macrophages.IMPORTANCEAlternaria species are ubiquitous environmental fungi to which the human host can continuously be exposed, through the inhalation of fungal spores but also of fragments of hyphae, from desegregated mycelia. These fungi are involved in hypersensitization and severe respiratory allergies, such as asthma, and can cause opportunistic infections in immunodepressed human host leading to severe disease. The first fungal structures to interact with the host cells are the cell wall components, and their modulation leads to differential immune responses. Here, we show that fungal cells grown with cell wall inhibitors led to cell wall nanoparticles with new properties in their interaction with macrophages. With this strategy, we overcame the limitation of in vitro assays interacting with filamentous fungi and showed that the absence of DNH-melanin leads to higher virulence, while caspofungin leads to cells walls that trigger higher hydrolysis of chitin and higher production of cytokines.

Exploring Antimicrobial Potency, ADMET, and Optimal Drug Target of a Non-ribosomal Peptide Sevadicin from Bacillus pumilus, through In Vitro Assay and Molecular Dynamics Simulation.

The current study was designed to explore the biosynthetic potential of sevadicin in Bacillus pumilus species and its interaction with bacterial drug target molecules. The non-ribosomal peptide (NRP) cluster in B. pumilus SF-4 was preliminarily confirmed using PCR-based screening, and the bioactivity of strain SF-4 culture extract was assessed against a set of human pathogenic strains. The susceptibility assay showed that strain SF-4 extract had higher inhibitory concentrations (312-375µg/mL) than ciprofloxacin. Genome mining of B. pumilus strains (n = 22) using AntiSMASH and BAGEL identified sevadicin coding biosynthetic gene cluster only in strain SF-4, constitutes of two core biosynthetic genes, three additional biosynthetic genes, two transport-related genes, and one regulatory gene. The molecular docking of sevadicin with various putative bacterial drug targets such as dihydropteroate, muramyl ligase E, topoisomerase, penicillin-binding protein, and in vitro safety analyses were conducted with detailed ADMET screening. The results showed that sevadicin makes hydrophobic interaction with MurE (PDB ID: 1E8C and 4C13) via hydrogen bonding, suggesting bacterial growth inhibition by disrupting the cell wall synthesis pathway and exhibiting a secure biosafety profile. The stability and compactness of sevadicin/MurE complexes were assessed via molecular dynamic simulation using RMSD, RMSF, and Rg. The simulation results revealed the binding stability of sevadicin/MurE complexes and indicated that the complexes can't be easily deformed. In conclusion, the current study explored the biosynthesis of sevadicin in B. pumilus for the first time and found that sevadicin inhibits bacterial growth by inhibiting cell wall synthesis via targeting the MurE enzyme and exhibits no toxicity.

A new therapeutic strategy for infectious diseases against intracellular multidrug-resistant bacteria

Bacterial infections result in 7,700,000 deaths per year globally, with intracellular bacteria causing repeated and resistant infection. No drug is currently licenced for the treatment of intracellular bacteria. A new screening platform mimicking the host milieu has been established to explore phytochemical antibiotic adjuvants. Previously neglected isoprenylated flavonoids were found to be effective against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Specifically, the synergistic effect between glabrol and streptomycin against intracellular bacteria was observed for the first time. The glabrol-streptomycin combination targets bacterial inner membrane phospholipids, disrupts arginine biosynthesis, inhibits cell wall proteins and biofilm formation genes (agrA/B/C/D), and promotes ROS production, causing subsequent membrane and wall damage.To enhance the selective uptake of combination drug into infected cells, hyaluronic acid-streptomycin-lipoic acid-glabrol nanoparticles (HSLGS-S) were designed and synthesized to trigger the intracellular delivery of the glabrol-streptomycin combination. Thus, the treatment can be transported into the infected intracellular region and selectively release the glabrol-streptomycin combination to the bacterial at site. The bioactivity of HSLGS-S in clearing intracellular bacteria was 20-fold higher than that of the glabrol-streptomycin combination alone in vitro and 2- to 10-fold higher in vivo.

Prevalence of the Cefazolin Inoculum Effect in Methicillin-Susceptible Staphylococcus aureus Isolates Collected from Kingston Health Sciences Centre.

Background: Staphylococcus aureus is a bacterium that causes serious human infections, including bloodstream infections, pneumonia, deep abscesses, and bone and joint infections. Methicillin-susceptible S. aureus (MSSA) are S. aureus bacteria susceptible to the antibiotic methicillin. Cefazolin is the first-line antibiotic to treat MSSA infections. The cefazolin inoculum effect (CzIE) is a lab phenomenon in which S. aureus is susceptible to cefazolin at a standard bacterial inoculum but develops resistance at a higher cell inoculum. CzIE occurs when the bacteria produce beta-lactamases that degrade cefazolin, preventing the antibiotic from inhibiting cell wall synthesis and allowing bacterial growth. In theory, CzIE may lead to cefazolin treatment failure, but currently, there is insufficient evidence in the literature. Objective: We aimed to determine the prevalence of the CzIE in patients with serious MSSA infections at Kingston Health Sciences Centre (KHSC). Methods: Clinical MSSA isolates from patients with serious infections at KHSC were collected. The MSSA isolates were then tested for CzIE using broth microdilution testing at a standard inoculum (5×10^5 CFU/mL) and a high inoculum (5×10^7 CFU/mL). The CzIE was defined as a ≥ 4-fold increase in minimum inhibitory concentration (MIC) from the standard to the high and with a MIC above the resistant breakpoint of 16 µg/mL. Results: Of the 51 MSSA isolates tested to date, 17 (33%) displayed CzIE, with 13 of the 17 (76%) requiring 64 µg/ml of antibiotic to prevent growth. Conclusion: Our initial testing indicates that the CzIE is common in MSSA infections at KHSC. We will complete testing 100 MSSA isolates for a more precise estimate of CzIE prevalence, which will be followed up with a chart review of the patients to determine if there is a correlation between CzIE and cefazolin treatment failure.

Phospho-code of a conserved transcriptional factor underpins fungal virulence

BackgroundCell wall integrity (CWI) is crucial for fungal growth, pathogenesis, and adaptation to extracellular environments. Calcofluor white (CFW) is a cell wall perturbant that inhibits fungal growth, yet little is known about how phytopathogenic fungi respond to the CFW-induced stress.ResultsIn this study, we unveiled a significant discovery that CFW triggered the translocation of the transcription factor CgCrzA from the cytoplasm to the nucleus in Colletotrichum gloeosporioides. This translocation was regulated by an interacting protein, CgMkk1, a mitogen-activated protein kinase involved in the CWI pathway. Further analysis revealed that CgMkk1 facilitated nuclear translocation by phosphorylating CgCrzA at the Ser280 residue. Using chromatin immunoprecipitation sequencing, we identified two downstream targets of CgCrzA, namely CgCHS5 and CgCHS6, which are critical for growth, cell wall integrity, and pathogenicity as chitin synthase genes.ConclusionsThese findings provide a novel insight into the regulatory mechanism of CgMkk1-CgCrzA-CgChs5/6, which enables response of the cell wall inhibitor CFW and facilitates infectious growth for C. gloeosporioides.

Calcium-Dependent Lipopeptide Antibiotics against Drug-Resistant Pathogens Discovered via Host-Dependent Heterologous Expression of a Cloned Biosynthetic Gene Cluster.

Historically, small molecules biosynthesised by bacteria have been an excellent source for antibacterial drugs. Today, however, the rediscovery of known compounds is a significant hurdle to developing new antimicrobials. Here we use a genome mining and synthetic biology approach to discover the ambocidins: calcium-dependent lipodepsipeptides that are active against drug-resistant Gram-positive pathogens. By cloning a silent biosynthetic gene cluster (the amb cluster) from Streptomyces ambofaciens ATCC 2387 and integrating this into the chromosome of Streptomyces avermitilis we induce expression of ambocidin A and B: two new Nε-hydroxyarginine-containing cyclic lipodepsipeptides active against drug-resistant Gram-positive pathogens. Using a panel of Streptomyces host strains, we show that the choice of heterologous host is critical for producing the biologically active compounds, and that inappropriate host choice leads to aberrant production inactive derivatives. We show that Nε-hydroxyarginine is the product of a haem-dependent oxygenase and that it enhances biological activity. Ambocidin A inhibits cell wall biosynthesis by binding to Lipid II at a different site than vancomycin. Unlike daptomycin, ambocidin A retains antimicrobial activity in the presence of lung-surfactant, giving it the potential to treatbacterial pneumonia. Our work expands the family of calcium-dependent lipopeptide antibiotics withnew members exhibiting a distinct mechanism of action.

Research progress on the effects of postharvest storage methods on melon quality

Background As an important global agricultural cash crop, melon has a long history of cultivation and a wide planting area. The physiological metabolism of melon after harvest is relatively strong; if not properly stored, melon is easily invaded by external pathogens during transportation, resulting in economic losses and greatly limiting its production, development and market supply. Therefore, the storage and freshness of melon are the main challenges in realizing the annual supply of melon, so postharvest storage has received increasing amounts of attention from researchers. Methods This study used academic, PubMed, and Web of Science resources to retrieve keywords related to postharvest storage and melon quality; read, refined, classified, and sorted the retrieved literature; sorted and summarized the relevant research results; and finally completed this article. Results This article reviews the mechanism and effects of physical, chemical and biological preservation techniques on the sensory quality, compound contents and respiratory physiological activities of different varieties of melon fruits. When maintaining normal metabolism and not producing physiological disorders, melon inhibits cell wall metabolism, reactive oxygen species metabolism and the ethylene biosynthesis pathway, etc., to the greatest extent during postharvest storage, thereby reducing the material consumption of fruits, delaying the ripening and senescence process, and prolonging the postharvest life and shelf life. Conclusion The literature provides a theoretical basis for postharvest preservation technology in the melon industry in the future and provides corresponding guidance for the development of the melon industry.

A rho-type GTPase activating protein affects the growth and development of Cordyceps cicadae.

Cordyceps cicadae is recognized for its medicinal properties, attributed to bioactive constituents like polysaccharides and adenosine, which have been shown to improve kidney and liver functions and possess anti-tumor properties. Rho GTPase activating proteins (Rho GAPs) serve as inhibitory regulators of Rho GTPases in eukaryotic cells by accelerating the GTP hydrolysis of Rho GTPases, leading to their inactivation. In this study, we explored the function of the CcRga8 gene in C. cicadae, which encodes a Rho-type GTPase activating protein. Our study found that the knockout of CcRga8 resulted in a decrease in polysaccharide levels and an increase in adenosine concentration. Furthermore, the mutants exhibited altered spore yield and morphology, fruiting body development, decreased infectivity, reduced resistance to hyperosmotic stress, oxidative conditions, and cell wall inhibitors. These findings suggest that CcRga8 plays a crucial role in the development, stress response, and bioactive compound production of C. cicadae.

Synergistic effects of sulopenem in combination with cefuroxime or durlobactam against Mycobacterium abscessus.

Treating infections from Mycobacterium abscessus (Mab), particularly those resistant to common antibiotics like macrolides, is notoriously difficult, akin to a never-ending struggle for healthcare providers. The rate of treatment failure is even higher than that seen with multidrug-resistant tuberculosis. The role of combination β-lactams in inhibiting L,D-transpeptidation, the major peptidoglycan crosslink reaction in Mab, is an area of intense investigation, and clinicians have utilized this approach in the treatment of macrolide-resistant Mab, with reports showing clinical success. In our study, we found that cefuroxime and sulopenem, when used together, display a significant synergistic effect. If this promising result seen in lab settings, translates well into real-world clinical effectiveness, it could revolutionize current treatment methods. This combination could either replace the need for more complex intravenous medications or serve as a "step down" to an oral medication regimen. Such a shift would be much easier for patients to manage, enhancing their comfort and likelihood of sticking to the treatment plan, which could lead to better outcomes in tackling these tough infections. Our research delved into how these drugs inhibit cell wall synthesis, examined time-kill data and binding studies, and provided a scientific basis for the observed synergy in cell-based assays.

Bacillus subtilis uses the SigM signaling pathway to prioritize the use of its lipid carrier for cell wall synthesis.

Peptidoglycan (PG) and most surface glycopolymers and their modifications are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP). These lipid-linked precursors are then flipped across the membrane and polymerized or directly transferred to surface polymers, lipids, or proteins. Despite its essential role in envelope biogenesis, UndP is maintained at low levels in the cytoplasmic membrane. The mechanisms by which bacteria distribute this limited resource among competing pathways is currently unknown. Here, we report that the Bacillus subtilis transcription factor SigM and its membrane-anchored anti-sigma factor respond to UndP levels and prioritize its use for the synthesis of the only essential surface polymer, the cell wall. Antibiotics that target virtually every step in PG synthesis activate SigM-directed gene expression, confounding identification of the signal and the logic of this stress-response pathway. Through systematic analyses, we discovered 2 distinct responses to these antibiotics. Drugs that trap UndP, UndP-linked intermediates, or precursors trigger SigM release from the membrane in <2 min, rapidly activating transcription. By contrasts, antibiotics that inhibited cell wall synthesis without directly affecting UndP induce SigM more slowly. We show that activation in the latter case can be explained by the accumulation of UndP-linked wall teichoic acid precursors that cannot be transferred to the PG due to the block in its synthesis. Furthermore, we report that reduction in UndP synthesis rapidly induces SigM, while increasing UndP production can dampen the SigM response. Finally, we show that SigM becomes essential for viability when the availability of UndP is restricted. Altogether, our data support a model in which the SigM pathway functions to homeostatically control UndP usage. When UndP levels are sufficiently high, the anti-sigma factor complex holds SigM inactive. When levels of UndP are reduced, SigM activates genes that increase flux through the PG synthesis pathway, boost UndP recycling, and liberate the lipid carrier from nonessential surface polymer pathways. Analogous homeostatic pathways that prioritize UndP usage are likely to be common in bacteria.

Near-freezing temperature suppresses avocado (Persea americana Mill.) fruit softening and chilling injury by maintaining cell wall and reactive oxygen species metabolism during storage

To enhance the postharvest quality of avocado (Persea americana Mill.) fruit, this study investigates alterations in cell wall metabolism and reactive oxygen species (ROS) metabolism during near-freezing temperature (NFT) storage, and explores their impact on fruit softening. The fruit was stored at 25 °C, 5 °C, 2 °C, and NFT, respectively. NFT storage retarded firmness loss and chilling injury in comparison with 25 °C, 5 °C, and 2 °C. NFT storage delayed the decrease of ionic-soluble pectin (ISP) and cellulose (CLL) contents by suppressing cell wall degradation enzyme activities. Correlation analysis showed that cell wall degradation enzyme activities were positively correlated to rates of ethylene release and respiration. Moreover, NFT storage maintained higher levels of DPPH and ABTS scavenging abilities, activities of superoxide dismutase, peroxidase, and catalase, as well as ascorbate-glutathione cycle (ascorbic acid, glutathione, glutathione disulfide, ascorbate peroxidase, cycle-related enzymes), thereby inhibited the increase of ROS content, malondialdehyde content, and cell membrane permeability. Fruit firmness and chilling injury were correlated with the contents of hydrogen (H2O2), superoxide anion (O2.-), ISP, and CLL. These results suggested that NFT could suppress fruit softening and chilling injury by inhibiting cell wall degradation through delaying respiration and ethylene production and suppressing ROS production via activation of antioxidant systems, thereby maintaining quality and prolonged storage life during avocado fruit storage.

Azetidines Kill Multidrug-Resistant Mycobacterium tuberculosis without Detectable Resistance by Blocking Mycolate Assembly.

Tuberculosis (TB) is the leading cause of global morbidity and mortality resulting from infectious disease, with over 10.6 million new cases and 1.4 million deaths in 2021. This global emergency is exacerbated by the emergence of multidrug-resistant MDR-TB and extensively drug-resistant XDR-TB; therefore, new drugs and new drug targets are urgently required. From a whole cell phenotypic screen, a series of azetidines derivatives termed BGAz, which elicit potent bactericidal activity with MIC99 values <10 μM against drug-sensitive Mycobacterium tuberculosis and MDR-TB, were identified. These compounds demonstrate no detectable drug resistance. The mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with cell envelope biogenesis, specifically late-stage mycolic acid biosynthesis. Transcriptomic analysis demonstrates that the BGAz compounds tested display a mode of action distinct from the existing mycobacterial cell wall inhibitors. In addition, the compounds tested exhibit toxicological and PK/PD profiles that pave the way for their development as antitubercular chemotherapies.

The Bacillus subtilis cell envelope stress-inducible ytpAB operon modulates membrane properties and contributes to bacitracin resistance.

Antibiotics that inhibit peptidoglycan synthesis trigger the activation of both specific and general protective responses. σM responds to diverse antibiotics that inhibit cell wall synthesis. Here, we demonstrate that cell wall-inhibiting drugs, such as bacitracin and cefuroxime, induce the σM-dependent ytpAB operon. YtpA is a predicted hydrolase previously proposed to generate the putative lysophospholipid antibiotic bacilysocin (lysophosphatidylglycerol), and YtpB is the branchpoint enzyme for the synthesis of membrane-localized C35 terpenoids. Using targeted lipidomics, we reveal that YtpA is not required for the production of lysophosphatidylglycerol. Nevertheless, ytpA was critical for growth in a mutant strain defective for homeoviscous adaptation due to a lack of genes for the synthesis of branched chain fatty acids and the Des phospholipid desaturase. Consistently, overexpression of ytpA increased membrane fluidity as monitored by fluorescence anisotropy. The ytpA gene contributes to bacitracin resistance in mutants additionally lacking the bceAB or bcrC genes, which directly mediate bacitracin resistance. These epistatic interactions support a model in which σM-dependent induction of the ytpAB operon helps cells tolerate bacitracin stress, either by facilitating the flipping of the undecaprenyl phosphate carrier lipid or by impacting the assembly or function of membrane-associated complexes involved in cell wall homeostasis.IMPORTANCEPeptidoglycan synthesis inhibitors include some of our most important antibiotics. In Bacillus subtilis, peptidoglycan synthesis inhibitors induce the σM regulon, which is critical for intrinsic antibiotic resistance. The σM-dependent ytpAB operon encodes a predicted hydrolase (YtpA) and the enzyme that initiates the synthesis of C35 terpenoids (YtpB). Our results suggest that YtpA is critical in cells defective in homeoviscous adaptation. Furthermore, we find that YtpA functions cooperatively with the BceAB and BcrC proteins in conferring intrinsic resistance to bacitracin, a peptide antibiotic that binds tightly to the undecaprenyl-pyrophosphate lipid carrier that sustains peptidoglycan synthesis.

The active ingredients in Chinese peony pods synergize with antibiotics to inhibit MRSA growth and biofilm formation

Staphylococcus aureus (S. aureus) is a zoonotic pathogen that infects both humans and animals. The rapid spread of methicillin-resistant S. aureus (MRSA) and its resistance to antibiotics, along with its ability to form biofilms, poses a serious challenge to the clinical application of traditional antibiotics. Peony (Paeonia lactiflora Pall.) is a traditional Chinese medicine with multiple pharmacological effects. This study observed the strong antibacterial and antibiofilm activity of the water extract (WE) and ethyl acetate extract (EA) of Chinese peony pods against MRSA. The combination of EA and vancomycin, cefotaxime, penicillin G or methicillin showed a synergistic or additive antibacterial and antibiofilm effects on MRSA, which is closely related to the interaction of 1,2,3,4,6-penta-O-galloyl-β-D-glucose (PG) and methyl gallate (MG). The active ingredients in peony pods have been found to increase the sensitivity of MRSA to antibiotics and demonstrate antibiofilm activity, which is mainly related to the down-regulation of global regulatory factors sarA and sigB, extracellular PIA and eDNA encoding genes icaA and cdiA, quorum sensing related genes agrA, luxS, rnaIII, hld, biofilm virulence genes psma and sspA, and genes encoding clotting factors coa and vwb, but is not related to genes that inhibit cell wall anchoring. In vivo test showed that both WE and EA were non-toxic and significantly prolonged the lifespan of G. mellonella larvae infected with MRSA. This study provides a theoretical basis for further exploration of the combined use of PG, MG and antibiotics to combat MRSA infections.

Comparative analyses of D-fenchone and MENA inhibited sprouting of sweet potato storage roots

Postharvest sprouting is a major problem for the storage of sweet potato. This study compared the effect of D-fenchone with methyl naphthacetate (MENA) treatments on the sprouting of ‘Yan 25′ sweet potato. The untreated control roots sprouted within 4 days however the roots fumigated with 200 μL / 23 L D-fenchone or MENA completely inhibited the sprouting of freshly harvested ‘Yan 25′ during 14 days storage at 30 ℃. Furthermore, treating established sprouts (0.5 cm) with MENA or D-fenchone resulted in damaged sprouts after seven days following treatment with higher MDA content and these sprouts eventually died. Transcriptomic analyses revealed that both D-fenchone and MENA inhibited cell wall synthesis and disrupted auxin and cytokinin pathways in the storage roots, by which suppressed the sprouting. The RNA-seq results of established sprouts showed that both D-fenchone and MENA promoted ethylene and reactive oxidative species (ROS) pathways, which were correlated with the physical damage. In addition, D-fenchone and MENA inhibited auxin pathway, MENA changed cytokinin and D-fenchone disrupted polyamine, cell division and cellulose biosynthesis pathways which all may have contributed to sprout suppression.

High productivity of immunostimulatory membrane vesicles of Limosilactobacillus antri using glycine.

Nanosized membrane vesicles (MVs) released by bacteria play important roles in both bacteria-bacteria and bacteria-host interactions. Some gram-positive lactic acid bacteria produce MVs exhibiting immunoregulatory activity in the host. We found that both bacterial cells and MVs of Limosilactobacillus antri JCM 15950, isolated from the human stomach mucosa, enhance immunoglobulin A production by murine Peyer's patch cells. However, the thick cell walls of gram-positive bacteria resulted in low MV production, limiting experiments and applications using MVs. In this study, we evaluated the effects of glycine, which inhibits cell wall synthesis, on the immunostimulatory MV productivity of L. antri. Glycine inhibited bacterial growth while increasing MV production, with 20 g/L glycine increasing MV production approximately 12-fold. Glycine was most effective at increasing MV production when added in the early exponential phase, which indicated that cell division in the presence of glycine increased MV production. Finally, glycine increased MV productivity approximately 16-fold. Furthermore, glycine-induced MVs promoted interleukin-6 production by macrophage-like J774.1 cells, and the immunostimulatory activity was comparable to that of spontaneously produced MVs. Our results indicate that glycine is an effective agent for improving the production of MVs with immunostimulatory activity in gram-positive lactic acid bacteria, which can be applied as mucosal adjuvants and functional foods.

Antimicrobial and anticancer properties of carbon monoxide releasing molecules of the fac-[Re(CO)3(N-N)L]+ family.

The toxicity profile of fac-[Re(CO)3(N-N)L]+ complexes against microbial and tumoral cells has been extensively studied, primarily focusing on modifications to the bidentate diimine (N-N) ligand. However, less attention has been paid to modifications of the axial ligand L, which is perpendicular to the Re-N-N plane. This study reveals that the high toxicity of the fac-[Re(CO)3(bpy)(Ctz)]+ complex may be attributed to the structural effect of the trityl (CPh3) group present in clotrimazole, as removal of phenyl rings causes a significant decrease in the activity against Staphylococcus aureus (S. aureus). Moreover, substitution of the 1-tritylimidazole ligand by the structurally related ligands PPh3 and PCy3 maintains similarly high activity levels. These findings contribute to understanding the interactions of toxic complexes with bacterial membranes, suggesting that the ligand structures play a crucial role in inhibiting cell wall synthesis processes, potentially including Lipid II synthesis. Compounds with Ph3E (E = C-imidazole; P) groups also showed to be 10 times more toxic than cisplatin against three mammalian cell lines (IC50: 2-4 μM). In contrast, the analogue 1-benzylimidazole and 1-tert-butylimidazole derivatives were as toxic as cisplatin. We observed that the decomposition of the [Re(I)(CO)3] fragment inside mammalian cell lines liberates CO, which is expected to exert biological effects. Therefore, compounds of this family possessing the structural motif Ph3E seem to combine high antimicrobial and antitumoral activities, the latter being much higher than that of cisplatin.