An antibiotic from an uncultured bacterium binds to an immutable target.

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, lipid II, and lipid IIIWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.

Characterization of the major autolysin (AtlC) of Staphylococcus carnosus.

BACKGROUND: Autolysis by cellular peptidoglycan hydrolases (PGH) is a well-known phenomenon in bacteria. During food fermentation, autolysis of starter cultures can exert an accelerating effect, as described in many studies on cheese ripening. In contrast, very little is known about autolysis of starter cultures used in other fermentations. Staphylococcus (S.) carnosus is often used in raw sausage fermentations, contributing to nitrate reduction and flavor formation. In this study, we analyzed the influence of PGHs of the strains S. carnosus TMW 2.146 and S. carnosus TMW 2.2525 on their autolytic behavior. The staphylococcal major autolysin (Atl), a bifunctional enzyme with an N-acetylmuramoyl-L-alanine amidase and a glucosaminidase as an active site, is assumed to be the enzyme by which autolysis is mainly mediated. RESULTS: AtlC mutant strains showed impaired growth and almost no autolysis compared to their respective wild-type strains. Light microscopy and scanning electron microscopy showed that the mutants could no longer appropriately separate from each other during cell division, resulting in the formation of cell clusters. The surface of the mutants appeared rough with an irregular morphology compared to the smooth cell surfaces of the wild-types. Moreover, zymograms showed that eight lytic bands of S. carnosus, with a molecular mass between 140 and 35 kDa, are processed intermediates of AtlC. It was noticed that additional bands were found that had not been described in detail before and that the banding pattern changes over time. Some bands disappear entirely, while others become stronger or are newly formed. This suggests that AtlC is degraded into smaller fragments over time. A second knockout was generated for the gene encoding a N-acetylmuramoyl-L-alanine amidase domain-containing protein. Still, no phenotypic differences could be detected in this mutant compared to the wild-type, implying that the autolytic activity of S. carnosus is mediated by AtlC. CONCLUSIONS: In this study, two knockout mutants of S. carnosus were generated. The atlC mutant showed a significantly altered phenotype compared to the wild-type, revealing AtlC as a key factor in staphylococcal autolysis. Furthermore, we show that Atl is degraded into smaller fragments, which are still cell wall lytic active.

Perfusion Pressure and the Histology of Brain Death: A Unique Case in an Infant Maintained on Life Support.

Brain death is a not uncommon phenomena in the adult and pediatric population. Most cases are removed from life support soon after brain death is declared. Less commonly, systemic perfusion is maintained by life support for some time after neurologic function stops. These cases present uncommon opportunities to explore the histology of necrosis and autolysis in the context of global hypoxic ischemic damage. Here, we describe the unusual case of an infant maintained on life support for 2 weeks after brain death was declared with an emphasis on the resulting gross and histologic findings including a discussion of their underlying physiology.

A molecular link between cell wall biosynthesis, translation fidelity, and stringent response in Streptococcus pneumoniae.

Survival in the human host requires bacteria to respond to unfavorable conditions. In the important Gram-positive pathogen Streptococcus pneumoniae, cell wall biosynthesis proteins MurM and MurN are tRNA-dependent amino acyl transferases which lead to the production of branched muropeptides. We demonstrate that wild-type cells experience optimal growth under mildly acidic stressed conditions, but ΔmurMN strain displays growth arrest and extensive lysis. Furthermore, these stress conditions compromise the efficiency with which alanyl-tRNAAla synthetase can avoid noncognate mischarging of tRNAAla with serine, which is toxic to cells. The observed growth defects are rescued by inhibition of the stringent response pathway or by overexpression of the editing domain of alanyl-tRNAAla synthetase that enables detoxification of tRNA misacylation. Furthermore, MurM can incorporate seryl groups from mischarged Seryl-tRNAAlaUGC into cell wall precursors with exquisite specificity. We conclude that MurM contributes to the fidelity of translation control and modulates the stress response by decreasing the pool of mischarged tRNAs. Finally, we show that enhanced lysis of ΔmurMN pneumococci is caused by LytA, and the murMN operon influences macrophage phagocytosis in a LytA-dependent manner. Thus, MurMN attenuates stress responses with consequences for host-pathogen interactions. Our data suggest a causal link between misaminoacylated tRNA accumulation and activation of the stringent response. In order to prevent potential corruption of translation, consumption of seryl-tRNAAla by MurM may represent a first line of defense. When this mechanism is overwhelmed or absent (ΔmurMN), the stringent response shuts down translation to avoid toxic generation of mistranslated/misfolded proteins.

Morphometric Indicators of Liver Acini of Deceased Newborns Depending on the Time of Death.

We studied morphometric changes in the liver acini of dead newborns depending on the duration of the postmortem period. Autopsy samples of the liver tissue from 49 dead newborns were divided into 7 groups depending on the time of death. Liver tissue samples were taken from the upper and lower areas of the liver in the supine position of newborns; paraffin sections were prepared and stained with hematoxylin and eosin. The morphometric analysis of histological preparations revealed a progressive decrease in the mean size of the liver plates (trabeculae) and, conversely, an increase in the area of sinusoids with increasing the duration of the postmortem period; these changes were due to the postmortem redistribution of the blood and autolysis processes. More significant changes were noted in acinar zone 3 of the lower part of the liver. The revealed intra-acinar features of postmortem changes should be taken into account for their differential diagnosis with pathological processes that developed during life, in particular, the signs of congestion and peliosis of the liver.

Morphometric Characteristics of Postmortem Changes in the Brain of Newborns Depending on the Duration of the Postmortem Period.

The morphometric characteristics of brain tissue were studied based on autopsy material from 49 deceased newborns divided into 7 groups based on the time after death. Samples were taken from the upper (frontal lobe) and lower (occipital lobe) regions relative to the supine position of the body. Paraffin sections were prepared from these samples and stained with hematoxylin and eosin. Histological preparations were analyzed using an image analysis system to determine the area of gliocyte nuclei, cross-cut blood vessels, and expanded area around them in the white matter of the brain. Based on these data, severity indices were calculated for both cellular (pericellular) and vascular (perivascular) expansion. The dynamics of changes in morphometric parameters within the range of cellular and vascular alterations on brain tissue specimens, which reflect the development of postmortem hypostasis and autolysis in this organ, can be used to estimate the duration of the postmortem interval. At the same time, these changes, when combined with other non-specific postmortem findings, should be distinguished from long-term pathological processes and diseases that may have occurred during life.

Mutation of mltG increases peptidoglycan fragment release, cell size, and antibiotic susceptibility in Neisseria gonorrhoeae.

IMPORTANCE: Neisseria gonorrhoeae is unusual in that the bacteria release larger amounts of cell wall material as they grow as compared to related bacteria, and the released cell wall fragments induce inflammation that leads to tissue damage in infected people. The study of MltG revealed the importance of this enzyme for controlling cell wall growth, cell wall fragment production, and bacterial cell size and suggests a role for MltG in a cell wall synthesis and degradation complex. The increased antibiotic sensitivities of mltG mutants suggest that an antimicrobial drug inhibiting MltG would be useful in combination therapy to restore the sensitivity of the bacteria to cell wall targeting antibiotics to which the bacteria are currently resistant.

Autolysis Affects the Iron Cargo of Ferritins in Neurons and Glial Cells at Different Rates in the Human Brain.

Iron is known to accumulate in neurological disorders, so a careful balance of the iron concentration is essential for healthy brain functioning. An imbalance in iron homeostasis could arise due to the dysfunction of proteins involved in iron homeostasis. Here, we focus on ferritin-the primary iron storage protein of the brain. In this study, we aimed to improve a method to measure ferritin-bound iron in the human post-mortem brain, and to discern its distribution in particular cell types and brain regions. Though it is known that glial cells and neurons differ in their ferritin concentration, the change in the number and distribution of iron-filled ferritin cores between different cell types during autolysis has not been revealed yet. Here, we show the cellular and region-wide distribution of ferritin in the human brain using state-of-the-art analytical electron microscopy. We validated the concentration of iron-filled ferritin cores to the absolute iron concentration measured by quantitative MRI and inductively coupled plasma mass spectrometry. We show that ferritins lose iron from their cores with the progression of autolysis whereas the overall iron concentrations were unaffected. Although the highest concentration of ferritin was found in glial cells, as the total ferritin concentration increased in a patient, ferritin accumulated more in neurons than in glial cells. Summed up, our findings point out the unique behaviour of neurons in storing iron during autolysis and explain the differences between the absolute iron concentrations and iron-filled ferritin in a cell-type-dependent manner in the human brain. The rate of loss of the iron-filled ferritin cores during autolysis is higher in neurons than in glial cells.

[Characteristics of histochemical, molecular genetic and radiation-induced liver changes depending on the postmortem interval]. / Kharakteristika gistokhimicheskikh, molekulyarno-geneticheskikh i luchevykh izmenenii pecheni v zavisimosti ot davnosti smerti.

The aim of this study is to analyze literature data on postmortem changes in the liver and their use in determination of postmortem interval. Biological death expectedly causes the development of postmortem disorders not only in the liver structure, but also changes in its biochemical and histochemical parameters. Literature data about changes of histochemical, immunohistochemical and biomolecular characteristics of liver tissue, as well as bacterial migration to the liver depending on the duration of postmortem period, are presented. The effectiveness of radiology for visualization of postmortem changes and, accordingly, for determining the postmortem interval is noted.

[Characteristics of structural morphological changes of the liver depending on the prescription of death coming]. / Kharakteristika strukturnykh morfologicheskikh izmenenii pecheni v zavisimosti ot davnosti smerti.

The purpose of the work is to analyze the literature data devoted to the study of postmortem morphological changes in liver tissue and their use to determine the prescription of death. Postmortem changes are based on the processes of postmortem redistribution of blood and autolysis, the speed and severity of development of which depends primarily on the lifetime pathology, as well as external temperature and humidity during storage of the corpse. The onset of biological death naturally entails the development of postmortem changes in the liver, manifested by a decrease in temperature, violations of the structure of organelles, cells and organ tissue as a whole. The determination and evaluation of developing postmortem morphological changes is necessary both for differential diagnosis with lifetime-developed pathological processes, and for determining the prescription of death coming. This necessitates research to study the features of the development of postmortem changes and to develop ways to assess them to determine the prescription of death coming.

A general strategy to inhibit serine protease by targeting its autolysis loop.

Serine proteases are a large family of enzymes critical for multiple physiological processes, and proven diagnostic and therapeutic targets in several clinical indications. The high similarity of active sites among different serine proteases posts a challenge to reach high selectivity for inhibitors of serine proteases targeting at the active site. Here, we demonstrated that one particular surface loop on serine proteases (autolysis loop) can be used to regulate their catalytic activity, through surveying the recent works including ours, and such an approach can reach high specificity. The autolysis loop is highly variable among different serine proteases, explaining the high specificity of inhibitors targeting the autolysis loop. We also outline the structural origin that links the perturbation of the autolysis loop and the inhibition of protease activity. Thus, the autolysis loop appears to be a highly sensitive allosteric site and can be used as a general handle to develop pharmacological agents to intervene with the activities of serine proteases in, eg, blood coagulation.

Transcriptomic analysis reveals process of autolysis of Kluyveromyces marxianus in vacuum negative pressure and the higher temperature.

Kluyveromyces marxianus is expected to be used in the production of yeast extracts due to its good fermentation ability and nutritional properties. Yeast autolysis is a key process to produce yeast extract and vacuum negative pressure stress can be used as an effective way to assist autolysis. However, the molecular mechanism of initiating Kluyveromyces marxianus autolysis induced by vacuum negative pressure and the higher temperature is still unclear. In this study, RNA-seq technology was performed mainly to analyze autolytic processes in Kluyveromyces marxianus strains. Considerable differentially expressed genes (DEGs) of downregulation were significantly enriched in 7 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to synthesis and transport of RNA and ribosome, which indicated that abnormal protein translations had already occurred in autolytic process. Interestingly, due to obvious change of related DEGs, endoplasmic reticulum-associated degradation (ERAD) and autophagy were activated and cell wall integrity pathway was hindered. Under the continuous influence of the external stress environment, the long-term changes of the above pathways triggered a vicious circle of gradual damage to yeast cells, which is the main cause of yeast autolysis. These results may provide important clues for the in-depth interpretation of the yeast autolytic mechanism.

Enhancing extracellular production of lipoxygenase in Escherichia coli by signal peptides and autolysis system.

BACKGROUND: Lipoxygenase (LOX) is a non-heme iron containing dioxygenase that is widely used to improve food quality and produce active drug intermediates and biodiesel. Escherichia coli is one of the most widely used host microorganisms for recombinant protein expression; however, its weak extracellular secretion ability precludes its effective production of recombinant proteins into the extracellular environment. To facilitate subsequent characterization and application of LOX, improving its secretion efficiency from E. coli is a major challenge that needs to be solved. RESULTS: Several strategies were adopted to improve the extracellular secretion of LOX based on the signal peptides and cell wall permeability of E. coli. Here, we studied the effect of signal peptides on LOX secretion, which increased the secretory capacity for LOX marginally. Although surfactants could increase the permeability of the cell membrane to promote LOX secretion, the extracellular LOX yield could not meet the requirements of industrialization production. Subsequently, an autolysis system was constructed in E. coli based on the bacteriophage lysis gene ΦX174-E to enhance the production of extracellular proteins. Thus, the extracellular production of LOX was achieved and the content of inclusion bodies in the cell was reduced by optimizing cell lysis conditions. The extracellular LOX yield reached 368 ± 1.4 U mL-1 in a 5-L bioreactor under optimized lysis conditions that is, an induction time and temperature, and arabinose concentration of 5 h, 25 °C, and 0.6 mM, respectively. CONCLUSIONS: In this study, the different signal peptides and cell autolysis system were developed and characterized for extracellular LOX production in E. coli. Finally, the cell autolysis system presented a slight advantage on extracellular LOX yield, which also provides reference for other protein extracellular production.

Production of autolysis-proof Kex2 protease from Candida albicans in Saccharomyces cerevisiae for in vitro processing of fusion proteins.

Protein expression with a fusion partner followed by the removal of the fusion partner via in vitro processing with a specific endoprotease is a favored method for the efficient production of intact recombinant proteins. Due to the high cost of commercial endoproteases, this process is restricted to laboratories. Kex2p is a membrane-bound serine protease that cleaves after dibasic residues of substrates in the late Golgi network. Although Kex2p is a very efficient endoprotease with exceptional specificity, it has not yet been used for the in vitro processing of fusion proteins due to its autolysis and high production cost. In this study, we developed an alternative endoprotease, autolysis-proof Kex2p, via site-directed mutagenesis of truncated KEX2 from Candida albicans (CaKEX2). Secretory production of manipulated CaKex2p was improved by employing target protein-specific translational fusion partner in Saccharomyces cerevisiae. The mass production of autolysis-proof Kex2p could facilitate the use of Kex2p for the large-scale production of recombinant proteins. KEY POINTS: • A soluble and active CaKex2p variant was produced by autocatalytic cleavage of the pro-peptide after truncation of C-terminus • Autolysis-proof CaKex2p was developed by site-directed mutagenesis • Secretion of autolysis-proof CaKex2p was improved by employing optimal translational fusion partner in Saccharomyces cerevisiae.

Evaluation of histological post-mortem changes in farmed Atlantic salmon (Salmo salar L.) at different time intervals and storage temperatures.

The aim of this study was to evaluate histologic post-mortem autolytic changes in farmed Atlantic salmon. The fish were either stored at room temperature (RT, 21°C), refrigerated (4°C) or frozen (-20°C), while fish necropsy was performed at 0, 1, 4, 24 and 48 h post-storage (hps). In addition, gills were sampled at 0, 5, 10, 15, 30 and 45 min post-storage (mps) at room temperature (RT). The haematoxylin and eosin-stained tissue slides were evaluated and scored by using a semi-quantitative scoring system. Our findings demonstrated gills and pyloric caeca/pancreas as the most severely autolysed organs while heart and skeletal musculature were least affected. Generally, moderate to severe autolysis appeared first at 4 hps, while severe changes were seen at 24 hps. Gills demonstrated autolytic changes as early as 10 mps and pyloric caeca/pancreas at 1 hps. Freezing did not prevent the autolysis and even contributed to freezing artefacts, which may lead to misdiagnosis. Keeping organs refrigerated slowed the autolytic progress within the first 4 hps marginally. This study recommends gills and pyloric caeca/pancreas should be sampled as early as possible, at least within 10 min post-necropsy.

A simple method for extracting phycocyanin from Arthrospira (Spirulina) platensis by autolysis.

Phycocyanin (PC) is a natural blue pigment that has great commercial value in food and pharmaceutical industry. Arthrospira (Spirulina) platensis is a photosynthetic spiral-shaped cyanobacterium containing a rich PC pigment. Autolysis is the enzymatic digestion of cells by the action of its own enzymes. To develop an effective and economical extraction process, an autolysis process was incorporated into the conventional freezing-thawing method. In the present study, 91% of maximal extraction yield of PC with 1.194 purity (A620/A280) was obtained via autolysis after 3 h of incubation at 37 °C without using an extraction salt solution or a successive freezing-thawing process. In addition to temperature, the initial concentration of bicarbonate in growth medium and the concentration of wet biomass are important parameters that influence the extraction yield of PC by autolysis.

Effects of Yeast Species and Processing on Intestinal Health and Transcriptomic Profiles of Atlantic Salmon (Salmo salar) Fed Soybean Meal-Based Diets in Seawater.

The objective of the current study was to examine the effects of yeasts on intestinal health and transcriptomic profiles from the distal intestine and spleen tissue of Atlantic salmon fed SBM-based diets in seawater. Cyberlindnera jadinii (CJ) and Wickerhamomyces anomalus (WA) yeasts were heat-inactivated with spray-drying (ICJ and IWA) or autolyzed at 50 °C for 16 h (ACJ and AWA), followed by spray-drying. Six diets were formulated, one based on fishmeal (FM), a challenging diet with 30% soybean meal (SBM) and four other diets containing 30% SBM and 10% of each of the four yeast fractions (i.e., ICJ, ACJ, IWA and AWA). The inclusion of CJ yeasts reduced the loss of enterocyte supranuclear vacuolization and reduced the population of CD8α labeled cells present in the lamina propria of fish fed the SBM diet. The CJ yeasts controlled the inflammatory responses of fish fed SBM through up-regulation of pathways related to wound healing and taurine metabolism. The WA yeasts dampened the inflammatory profile of fish fed SBM through down-regulation of pathways related to toll-like receptor signaling, C-lectin receptor, cytokine receptor and signal transduction. This study suggests that the yeast species, Cyberlindnera jadinii and Wickerhamomyces anomalus are novel high-quality protein sources with health-beneficial effects in terms of reducing inflammation associated with feeding plant-based diets to Atlantic salmon.

The exogenous autophagy inducement alleviated the sea cucumber (Stichopus japonicus) autolysis with exposure to stress stimuli of ultraviolet light.

BACKGROUND: Autolysis is the most important restrictive factor for the live sea cucumber trade and commercial transportation. Thus, it is essential to investigate the mechanism of autolysis activation or deactivation in the sea cucumber. In this study, monodansylcadaverine staining and Western blotting experiment methods indicated the implication of autophagy in the ultraviolet (UV) exposed sea cucumbers. The health condition was observed after the sea cucumbers (Stichopus japonicus) were gastric perfusion with autophagic inhibitor (3-methyladenine) or inducer (rapamycin) and exposure to UV light for half an hour. RESULTS: The protein expressions of LC3-II and Atg5 appeared immediately after UV exposure and then vanished 1 h later. The autophagosome formation in coelomic fluid cells confirmed the autophagy appearance pattern of LC3-II and Atg5. The sea cucumber individuals maintained the health condition during the entire event of autophagy. The autophagic inhibitor along with UV exposure contributed to sea cucumber's swollen intestinal tissues, but the autophagic inducer functioned to alleviate and neutralize the UV effect. CONCLUSIONS: The autophagy procedure analysis demonstrated that autophagy plays a role to maintain the health condition of sea cucumber during autolysis inducement. The autolysis of sea cucumber can be alleviated or postponed by the exogenous autophagy inducer and this finding would benefit the live sea cucumber transportation. © 2021 Society of Chemical Industry.

Protein hydrolysates derived from aquaculture and marine byproducts through autolytic hydrolysis.

Autolysis technology has shown potential for protein hydrolysates production from marine and aquaculture byproducts. Viscera are a source of cheap proteolytic enzymes for producing protein hydrolysates from the whole fish or processing byproducts of the most valuable commercial species by applying autolysis technology. The use of autolysis allows economical production of protein hydrolysate and provides an opportunity to valorize downstream fish and shellfish processing byproducts at a lower cost. As a result, production and application of marine byproduct autolysates is increasing in the global protein hydrolysates market. Nevertheless, several restrictions occur with autolysis, including lipid and protein oxidation mediated by the heterogeneous composition of byproducts. The generally poor storage and handling of byproducts may increase the formation of undesirable metabolites during autolysis, which can be harmful. The formation of nitrogenous compounds (i.e., biogenic amines), loss of freshness, and process of autolysis in the byproducts could increase the rate of quality and safety loss and lead to more significant concern about the use of autolysates for human food applications. The current review focuses on the autolysis process, which is applied for the hydrolysis of aquaculture and marine discards to obtain peptides as functional or nutritive ingredients. It further addresses the latest findings on the mechanisms and factors contributing the deterioration of byproducts and possible ways to control oxidation and other food quality and safety issues in raw materials and protein hydrolysates.

Effect of the cold pre-fermentative maceration and aging on lees times on the phenolic compound profile, antioxidant capacity and color of red sparkling wines.

This was the first study evaluating the impact of cold pre-fermentative maceration using refrigeration on the nutraceutical quality and color of red sparkling wines elaborated with the cultivar Syrah, and the evolution of these variables with different autolysis times. The sparkling wines were elaborated using the traditional method with different maceration times (NM, 24 and 72 h) and aging on lees (3 and 18 months of autolysis). In the sequence, it was conducted the characterization of the phenolic compound profile by HPLC-DAD (n = 21), the antioxidant capacity (ABTS, DPPH, and FRAP assays), and the color (CIELab and CIEL*C*h systems). The total phenolic content (TPC) and antioxidant capacity (AOX) were higher with longer maceration (M72) and autolysis (18 months) times, reaching 453.54 mg L-1 of TPC, and AOX above 2.11 mmol TEAC L-1 by the three in vitro assays conducted. Cis-resveratrol, kaempferol-3-O-glucoside, quercetin-3-ß-d-glucoside, isorhamnetin-3-O-glucoside, and petunidin-3-O-glucoside showed a good correlation (r > 0.8; P < 0.05) with the antioxidant capacity and were found in higher concentrations in the sparkling wines elaborated with maceration. In addition, maceration promoted a more intense red (a*) and saturated (C*) color. Thus, the results indicated that cold pre-fermentative maceration and autolysis positively influenced the bioactive potential and the color of the red sparkling wines. This practice should be better explored through the elaboration of this product. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05531-z.