-
Microbes and Infection Mar 2014Streptococcus pneumoniae (SP) and nontypeable Haemophilus influenzae (NTHi) are common commensals of the human airway and major bacterial pathogens of otitis media (OM)...
Streptococcus pneumoniae (SP) and nontypeable Haemophilus influenzae (NTHi) are common commensals of the human airway and major bacterial pathogens of otitis media (OM) and other upper airway infections. The interaction between them may play an important role in the pathogenesis of polymicrobial infections. Although previous studies suggested NTHi could promote pneumococcal survival and biofilm formation, how NTHi affects pneumococcal activities has not been defined. Our data in the present studies indicated that the outcome of the interaction between SP and NTHi was in a cell-density-dependent manner and the enhancement of pneumococcal survival happened at the later stages of culturing. Using quantitative PCR, we found that the expression of pneumococcal genes regulating autolysis and fratricide, lytA and cbpD, were significantly down-regulated in co-culture with NTHi. We further observed that influence of NTHi was not on direct cell-to-cell contact, but that this contact may contribute to the interaction between these two microorganisms. These results suggest that pneumococcal survival and biofilm formation can be enhanced by down-regulating pneumococcal cell wall hydrolase production thereby inhibiting pneumococcal autolysis and fratricide in the presence of NTHi.
Topics: Bacteriolysis; Biofilms; Coculture Techniques; Haemophilus influenzae; Microbial Interactions; Microbial Viability; Streptococcus pneumoniae
PubMed: 24269704
DOI: 10.1016/j.micinf.2013.11.006 -
Scientific Reports Sep 2020Streptococcus pneumoniae choline kinase (sChoK) has previously been proposed as a drug target, yet the effectiveness of the first and only known inhibitor of sChoK,...
Streptococcus pneumoniae choline kinase (sChoK) has previously been proposed as a drug target, yet the effectiveness of the first and only known inhibitor of sChoK, HC-3, is in the millimolar range. The aim of this study was thus to further validate sChoK as a potential therapeutic target by discovering more powerful sChoK inhibitors. LDH/PK and colorimetric enzymatic assays revealed two promising sChoK inhibitor leads RSM-932A and MN58b that were discovered with IC50 of 0.5 and 150 μM, respectively, and were shown to be 2-4 magnitudes more potent than the previously discovered inhibitor HC-3. Culture assays showed that the minimum inhibitory concentration (MIC) of RSM-932A and MN58b for S. pneumoniae was 0.4 μM and 10 μM, respectively, and the minimum lethal concentration (MLC) was 1.6 μM and 20 μM, respectively. Western blot monitoring of teichoic acid production revealed differential patterns in response to each inhibitor. In addition, both inhibitors possessed a bacteriostatic mechanism of action, and neither interfered with the autolytic effects of vancomycin. Cells treated with MN58b but not RSM-932A were more sensitive to a phosphate induced autolysis with respect to the untreated cells. SEM studies revealed that MN58b distorted the cell wall, a result consistent with the apparent teichoic acid changes. Two novel and more highly potent putative inhibitors of sChoK, MN58b and RSM-932A, were characterized in this study. However, the effects of sChoK inhibitors can vary at the cellular level. sChoK inhibition is a promising avenue to follow in the development of therapeutics for treatment of S. pneumoniae.
Topics: Aniline Compounds; Autolysis; Butanes; Cell Wall; Choline Kinase; Microbial Sensitivity Tests; Protein Kinase Inhibitors; Pyridinium Compounds; Quinolinium Compounds; Streptococcus pneumoniae; Teichoic Acids
PubMed: 32963303
DOI: 10.1038/s41598-020-72165-6 -
Nature Communications Mar 2016Biogenesis of the 20S proteasome is tightly regulated. The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion...
Biogenesis of the 20S proteasome is tightly regulated. The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease.
Topics: Aspartic Acid; Autolysis; Catalysis; Catalytic Domain; Crystallography, X-Ray; Cysteine; Lysine; Mutagenesis, Site-Directed; Proteasome Endopeptidase Complex; Protein Precursors; Proteolysis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Serine; Threonine
PubMed: 26964885
DOI: 10.1038/ncomms10900 -
FEMS Microbiology Ecology Apr 2015The amounts of carbon allocated to the fungal partner in ectomycorrhizal associations can vary substantially depending on the plant growth and the soil nutrient...
The amounts of carbon allocated to the fungal partner in ectomycorrhizal associations can vary substantially depending on the plant growth and the soil nutrient conditions, and the fungus may frequently be confronted with limitations in carbon. We used chemical analysis and transcriptome profiling to examine the physiological response of the ectomycorrhizal fungus Paxillus involutus to carbon starvation during axenic cultivation. Carbon starvation induced a decrease in the biomass. Concomitantly, ammonium, cell wall material (chitin) and proteolytic enzymes were released into the medium, which suggest autolysis. Compared with the transcriptome of actively growing hyphae, about 45% of the transcripts analyzed were differentially regulated during C-starvation. Induced during starvation were transcripts encoding extracellular enzymes such as peptidases, chitinases and laccases. In parallel, transcripts of N-transporters were upregulated, which suggest that some of the released nitrogen compounds were re-assimilated by the mycelium. The observed changes suggest that the carbon starvation response in P. involutus is associated with complex cellular changes that involves autolysis, recycling of intracellular compounds by autophagy and reabsorption of the extracellular released material. The study provides molecular markers that can be used to examine the role of autolysis for the turnover and survival of the ectomycorrhizal mycelium in soils.
Topics: Autolysis; Basidiomycota; Biomass; Carbon; Carrier Proteins; Chitinases; Gene Expression Profiling; Hyphae; Laccase; Mycelium; Mycorrhizae; Peptide Hydrolases; Soil; Soil Microbiology; Transcriptome
PubMed: 25778509
DOI: 10.1093/femsec/fiv027 -
The Plant Journal : For Cell and... Oct 2008Establishing the mechanisms regulating the autolysis of xylem tracheary elements (TEs) is important for understanding this programmed cell death process. These data...
Establishing the mechanisms regulating the autolysis of xylem tracheary elements (TEs) is important for understanding this programmed cell death process. These data demonstrate that two paralogous Arabidopsis thaliana proteases, XYLEM CYSTEINE PROTEASE1 (XCP1) and XCP2, participated in micro-autolysis within the intact central vacuole before mega-autolysis was initiated by tonoplast implosion. The data acquisition was aided by the predictable pattern of seedling root xylogenesis, the availability of single and double total knock-out T-DNA lines, anti-sera that recognized XCP1 and XCP2, and the microwave-assisted processing of whole seedlings prior to immunolabeling and observation in the transmission electron microscope. During secondary wall thickening, XCP1 and XCP2 (in wild type), XCP1 (in xcp2 seedlings) or XCP2 (in xcp1 seedlings) were imported into the TE central vacuole. Both XCP1 and XCP2 heavily labeled dense aggregates of material within the vacuole. However, because of XCP1 deficiency in xcp1 and xcp1 xcp2 TEs, non-degraded cellular remnants first accumulated in the vacuole and then persisted in the TE lumen (longer than in the wild type) after the final mega-autolysis was otherwise complete. This delayed TE clearing phenotype in xcp1 was rescued by complementation with wild-type XCP1. Although TEs in the xcp2 single knock-out cleared comparably with wild type, the non-degraded remnants in xcp1 xcp2 TEs were more densely packed than in xcp1 TEs. Therefore, XCP2 has a minor but distinct role in micro-autolysis. After tonoplast implosion, XCP1 and XCP2 remained associated with disintegrating cellular material as mega-autolysis, aided by additional lytic enzymes, destroyed the bulk of the cellular contents.
Topics: Arabidopsis; Arabidopsis Proteins; Cysteine Endopeptidases; DNA, Bacterial; DNA, Plant; Genes, Plant; Genetic Complementation Test; Genotype; Immunoblotting; Microscopy, Electron, Transmission; Mutagenesis, Insertional; Plant Roots; Vacuoles; Xylem
PubMed: 18573193
DOI: 10.1111/j.1365-313X.2008.03592.x -
Journal of Bacteriology Apr 1980Autolysis of unwashed exponential-phase Escherichia coli cells was efficiently promoted by first submitting them to a quick downshock with distilled water before an...
Autolysis of unwashed exponential-phase Escherichia coli cells was efficiently promoted by first submitting them to a quick downshock with distilled water before an upshock with 0.5 M sodium acetate, pH 6.5. The association of these two osmotic shocks had a remarkable synergistic effect and led to significant decreases in turbidity and viability. Different factors influencing the rate of cell lysis were examined. A close correlation was established between autolysis and the degradation of peptidoglycan. Both phenomena were induced by the same shock treatment, followed similar kinetics, and were efficiently blocked by addition of divalent cations. Cell lysis was also inducible by a shock treatment with 10(-3) M ethylenediaminetetraacetic acid or ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid and blocked by the addition of divalent cations.
Topics: Bacteriolysis; Buffers; Cations; Edetic Acid; Egtazic Acid; Escherichia coli; Hydrogen-Ion Concentration; Osmotic Pressure; Peptidoglycan; Temperature
PubMed: 6768717
DOI: 10.1128/jb.142.1.52-59.1980 -
Heliyon Mar 2024Fish protein hydrolysates were obtained from cultured rainbow trout (Oncorhynchus mykiss) viscera using commercial and endogenous enzymes. Two methods were employed for...
Fish protein hydrolysates were obtained from cultured rainbow trout (Oncorhynchus mykiss) viscera using commercial and endogenous enzymes. Two methods were employed for hydrolysis: acid autolysis (also known as silage) at room temperature for 10 days in acidic conditions, until total solubilisation, and enzymatic hydrolysis using Alcalase 2.4 LFG, Protana Prime, and the endogenous enzymes in the viscera. The effectiveness of both methods in releasing free amino acids (FAA) was assessed. After evaluating the results, the most effective enzymatic hydrolysis was optimized. The findings indicated that enzymatic hydrolysis with Alcalase, Protana Prime and endogenous enzymes combined for 7 h at a dose of 1% of protein, and a 7-day acid autolysis yielded the highest degree of hydrolysis (83.8% and 75.8%), a yield of FAA from viscera of 5.9% and 3.2%, and a yield of FAA from total protein of 71.3% and 52.5%, respectively. In conclusion, the use of commercial enzymes was more efficient in releasing amino acids, but endogenous enzymes showed a strong proteolytic capacity during acid autolysis, suggesting it also as a promising method to produce FAA-rich hydrolysates.
PubMed: 38468971
DOI: 10.1016/j.heliyon.2024.e27030 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Apr 2019Yeast autolysis under solid-state fermentation can effectively promote the release of various active substances, thereby improving the quality of yeast products. The...
Yeast autolysis under solid-state fermentation can effectively promote the release of various active substances, thereby improving the quality of yeast products. The optimal process for yeast autolysis under solid-state fermentation was obtained by optimizing the autolysis temperature, autolysis time and the zinc ion concentration. We analyzed the indexes of free amino acid, soluble protein and α-amino nitrogen in the fermentation material, as well as A₂₆₀/A₂₈₀ ratio to determine yeast autolysis process conditions in the solid-state fermentation. On the basis of the obtained data, L₉ (3³) orthogonal test was designed to optimize the solid-state fermentation parameters for yeast autolysis: temperature at 40, 50 and 55 °C; time 12, 18 and 24 h; zinc ion concentration 2, 4 and 8 mg/kg. The optimum process conditions for yeast autolysis were: autolysis temperature 55 °C, time 18 h, zinc ion concentration 2 mg/kg, and soluble protein content reached 9.31 mg/g, free amino acid 14.36 mg/g, α-amino nitrogen 10.16 μg/g and A₂₆₀/A₂₈₀ 1.73. After optimization of the process, the soluble protein, free amino acid and α-amino nitrogen contents of the yeast autolysis production can be significantly increased, thereby obviously improving the quality of the composite culture.
Topics: Amino Acids; Autolysis; Fermentation; Humans; Hydrogen-Ion Concentration; Nitrogen; Saccharomyces cerevisiae; Temperature
PubMed: 31001958
DOI: 10.13345/j.cjb.180341 -
PloS One 2014Autolysis of lactic acid bacteria (LAB) plays a vital role in dairy processing. During cheese making, autolysis of LAB affects cheese flavor development through release...
Autolysis of lactic acid bacteria (LAB) plays a vital role in dairy processing. During cheese making, autolysis of LAB affects cheese flavor development through release of intracellular enzymes and restricts the proliferation of cells in yogurt fermentation and probiotics production. In order to explore the mechanism of autolysis, the gene for the autolytic enzymes of L. bulgaricus, N-acetylmuramidase (mur), was cloned and sequenced (GenBank accession number: KF157911). Mur gene overexpression and gene knockout vectors were constructed based on pMG76e and pUC19 vectors. Recombinant plasmids were transformed into L. bulgaricus ljj-6 by electroporation, then three engineered strains with pMG76e-mur vector and fifteen engineered strains with pUC19-mur::EryBII were screened. The autolysis of the mur knockout strain was significantly lower and autolysis of the mur overexpressed strain was significantly higher compared with that of the wild type strain ljj-6. This result suggested that the mur gene played an important role in autolysis of L. bulgaricus. On the other hand, autolytic activity in a low degree was still observed in the mur knockout strain, which implied that other enzymes but autolysin encoded by mur were also involved in autolysis of L. bulgaricus.
Topics: Autolysis; Base Sequence; Cheese; Cloning, Molecular; DNA, Bacterial; Fermentation; Gene Knockout Techniques; Glycoside Hydrolases; Lactobacillus delbrueckii; Molecular Sequence Data; Sequence Analysis, DNA; Yogurt
PubMed: 25110891
DOI: 10.1371/journal.pone.0104829 -
Plant Physiology May 1991Cell walls isolated from auxin-pretreated maize (Zea mays L.) coleoptile segments were assayed to disclose evidence for the existence of enhanced autolysis. To improve...
Cell walls isolated from auxin-pretreated maize (Zea mays L.) coleoptile segments were assayed to disclose evidence for the existence of enhanced autolysis. To improve the sensitivity of the measurements and to facilitate kinetic analysis, isolated cell walls were consolidated within a small column, and the autolysis rate was directly determined from the sugar content of the effluent. This protocol revealed that the maximum rate of autohydrolysis of walls prepared from segments occurs within the first 2 hours and a steady decline commences almost immediately. Walls from indoleacetic acid pretreated segments (0.5-4 hours) released sugar at a higher rate initially (110-125% of controls) and the enhanced rate of autolysis continued for 6 to 8 hours, but then it became equivalent to that of the controls. Pretreatment of the segments at acidic pH had no effect on the measurable rates of autolysis. The (1-->3), (1-->4)-beta-d-glucan content of the walls and the extractable glucanase activities support the hypothesis that temporal enhancement of autohydrolysis is a function of auxin on enzyme activity. The progressive decline in autolysis during prolonged incubations is consistent with the decrease in the quantity of the beta-d-glucan in the wall. The relationship between glucan content and autolysis rate is supported by the observation that while glucose pretreatment of segments had only a small effect on initial autolysis rates, the presence of the sugar during pretreatment served to extend the interval over which higher rates of autolysis could be sustained. The results demonstrate that autolysis is related to auxin-induced wall metabolism in maize coleoptiles.
PubMed: 16668166
DOI: 10.1104/pp.96.1.285