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Journal of Microbiology and... Apr 2024Microorganisms usually coexist as a multifaceted polymicrobial community in the natural habitats and at mucosal sites of the human body. Two opportunistic human...
Microorganisms usually coexist as a multifaceted polymicrobial community in the natural habitats and at mucosal sites of the human body. Two opportunistic human pathogens, and commonly coexist in the bacterial infections for hospitalized and/or immunocompromised patients. Here, we observed that autolysis of the quorum-sensing (QS) mutant () was suppressed by the presence of the cells in vitro. The QS mutant still displayed killing against cells, suggesting the link between the -killing activity and the autolysis suppression. Independent screens of the transposon mutants defective in the -killing and the transposon mutants devoid of the autolysis suppression revealed the genetic link between both phenotypes, suggesting that the iron-dependent metabolism involving exoproteins might be central to both phenotypes. The autolysis was suppressed by iron treatment as well. These results suggest that the interaction between and might be governed by mechanisms that necessitate the QS circuitry as well as the metabolism involving the extracellular iron resources during the polymicrobial infections in the human airway.
Topics: Pseudomonas aeruginosa; Quorum Sensing; Staphylococcus aureus; Iron; Mutation; Bacterial Proteins; Humans; Bacteriolysis; Microbial Interactions; DNA Transposable Elements
PubMed: 38303126
DOI: 10.4014/jmb.2312.12028 -
Applied Biochemistry and Biotechnology Mar 2024Staphylococcus aureus is a major cause of hospital-associated infections worldwide. The organism's ability to form biofilms has led to resistance against current...
Staphylococcus aureus is a major cause of hospital-associated infections worldwide. The organism's ability to form biofilms has led to resistance against current treatment options such as beta-lactams, glycopeptides, and daptomycin. The ArlRS two-component system is a crucial regulatory system necessary for S. aureus autolysis, biofilm formation, capsule synthesis, and virulence. This study aims to investigate the role of the arlR deletion mutant in the detection and activation of S. aureus. We created an arlR deleted mutant and complementary strains and characterized their impact on the strains using partial growth measurement. The quantitative real-time PCR was performed to determine the expression of icaA, and the microscopic images of adherent cells were captured at the optical density of 600 to determine the primary bacterial adhesion. The biofilm formation assay was utilized to investigate the number of adherent cells using crystal violet staining. Eventually, the Triton X-100 autolysis assay was used to determine the influence of arlR on the cell autolytic activities. Our findings indicate that the deletion of arlR reduced the transcriptional expression of icaA but not icaR in the ica operon, leading to decrease in polysaccharide intercellular adhesin (PIA) synthesis. Compared to the wild-type and the complementary mutants, the arlR mutant exhibited decreased in biofilm production but increased autolysis. It concluded that the S. aureus response regulatory ArlR influences biofilm formation, agglutination, and autolysis. This work has significantly expanded our knowledge of the ArlRS two-component regulatory system and could aid in the development of novel antimicrobial strategies against S. aureus.
PubMed: 38530540
DOI: 10.1007/s12010-024-04919-1 -
Frontiers in Cellular and Infection... 2024forms biofilms consisting of cells embedded in a matrix made of proteins, polysaccharides, lipids, and extracellular DNA (eDNA). Biofilm-associated infections are... (Review)
Review
forms biofilms consisting of cells embedded in a matrix made of proteins, polysaccharides, lipids, and extracellular DNA (eDNA). Biofilm-associated infections are difficult to treat and can promote antibiotic resistance, resulting in negative healthcare outcomes. eDNA within the matrix contributes to the stability, growth, and immune-evasive properties of biofilms. eDNA is released by autolysis, which is mediated by murein hydrolases that access the cell wall via membrane pores formed by holin-like proteins. The eDNA content of biofilms varies among individual strains and is influenced by environmental conditions, including the presence of antibiotics. eDNA plays an important role in biofilm development and structure by acting as an electrostatic net that facilitates protein-cell and cell-cell interactions. Because of eDNA's structural importance in biofilms and its ubiquitous presence among isolates, it is a potential target for therapeutics. Treatment of biofilms with DNase can eradicate or drastically reduce them in size. Additionally, antibodies that target DNABII proteins, which bind to and stabilize eDNA, can also disperse biofilms. This review discusses the recent literature on the release, structure, and function of eDNA in biofilms, in addition to a discussion of potential avenues for targeting eDNA for biofilm eradication.
Topics: Biofilms; Staphylococcus aureus; DNA, Bacterial; Staphylococcal Infections; Humans; Bacterial Proteins; Anti-Bacterial Agents
PubMed: 38903938
DOI: 10.3389/fcimb.2024.1400648 -
Biochimica Et Biophysica Acta. General... Jan 2024Ischemia and reperfusion (I/R) injury exacerbate the prognosis of ischemic diseases. The cause of this exacerbation is partly a mitochondrial cell death pathway....
BACKGROUND
Ischemia and reperfusion (I/R) injury exacerbate the prognosis of ischemic diseases. The cause of this exacerbation is partly a mitochondrial cell death pathway. Mitochondrial calpain-5 is proteolyzed/autolyzed under endoplasmic reticulum stress, resulting in inflammatory caspase-4 activation. However, the role of calpain-5 in I/R injury remains unclear. We hypothesized that calpain-5 is involved in ischemic brain disease.
METHODS
Mitochondria from C57BL/6J mice were extracted via centrifugation with/without proteinase K treatment. The expression and proteolysis/autolysis of calpain-5 were determined using western blotting. The mouse and human brains with I/R injury were analyzed using hematoxylin and eosin staining and immunohistochemistry. HT22 cells were treated with tunicamycin and CAPN5 siRNA.
RESULTS
Calpain-5 was expressed in the mitochondria of mouse tissues. Mitochondrial calpain-5 in mouse brains was responsive to calcium earlier than cytosolic calpain-5 in vitro calcium assays and in vivo bilateral common carotid artery occlusion model mice. Immunohistochemistry revealed that neurons were positive for calpain-5 in the normal brains of mice and humans. The expression of calpain-5 was increased in reactive astrocytes at human infarction sites. The knockdown of calpain-5 suppressed of cleaved caspase-11.
CONCLUSIONS
The neurons of human and mouse brains express calpain-5, which is proteolyzed/autolyzed in the mitochondria in the early stage of I/R injury and upregulated in reactive astrocytes in the end-stage.
GENERAL SIGNIFICANCE
Our results provide a comprehensive understanding of the mechanisms underlying I/R injury. Targeting the expression or activity of mitochondrial calpain-5 may suppress the inflammation during I/R injuries such as cerebrovascular diseases.
Topics: Animals; Mice; Humans; Calpain; Calcium; Mice, Inbred C57BL; Brain Ischemia; Reperfusion Injury; Caspases
PubMed: 37949151
DOI: 10.1016/j.bbagen.2023.130506 -
International Journal of Biological... Dec 2023Bacillus amyloliquefaciens LB1ba02 is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. However, autolysis...
Bacillus amyloliquefaciens LB1ba02 is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. However, autolysis affects the growth of bacteria, further affecting the yield of target products. Besides, the restriction-modification system, existed in B. amyloliquefaciens LB1ba02, results in a low transformation efficiency, which further leads to a lack of high-throughput screening tools. Here, we constructed a genome-wide crRNA inhibition library based on the CRISPR/dCpf1 system and high-throughput screening of related genes affecting the cell growth and autolysis using flow cytometry in B. amyloliquefaciens LB1ba02. The whole genome crRNA library was first validated for resistance to the toxic chemical 5-fluorouracil, and then used for validation of essential genes. In addition, seven gene loci (oppD, flil, tuaA, prmA, sigO, hslU, and GE03231) that affect the growth characteristics of LB1ba02 were screened. Among them, the Opp system had the greatest impact on growth. When the expression of operon oppA-oppB-oppC-oppD-oppF was inhibited, the cell growth difference was most significant. Inhibition of other sites could also promote rapid growth of bacteria to varying degrees; however, inhibition of GE03231 site accelerated cell autolysis. Therefore, the whole genome crRNA inhibition library is well suited for B. amyloliquefaciens LB1ba02 and can be further applied to high-throughput mining of other functional genes.
Topics: Bacillus amyloliquefaciens; RNA, Guide, CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; High-Throughput Screening Assays; Operon
PubMed: 37802457
DOI: 10.1016/j.ijbiomac.2023.127179 -
BioRxiv : the Preprint Server For... Jul 2023Most bacterial cell envelopes contain a cell wall layer made of peptidoglycan. The synthesis of new peptidoglycan is critical for cell growth, division and...
UNLABELLED
Most bacterial cell envelopes contain a cell wall layer made of peptidoglycan. The synthesis of new peptidoglycan is critical for cell growth, division and morphogenesis, and is also coordinated with peptidoglycan hydrolysis to accommodate the new material. However, the enzymes that cleave peptidoglycan must be carefully controlled to avoid autolysis. In recent years, some control mechanisms have begun to emerge, although there are many more questions than answers for how most cell wall hydrolases are regulated. Here, we report a novel cell wall hydrolase control mechanism in , which we discovered during our characterization of a mutant sensitive to the overproduction of a secretin protein. The mutation affected an uncharacterized Sel1-like repeat protein encoded by the PA3978 locus. In addition to the secretin-sensitivity phenotype, PA3978 disruption also increased resistance to a β-lactam antibiotic used in the clinic. and analysis revealed that PA3978 binds to the catalytic domain of the lytic transglycosylase MltF and inhibits its activity. ΔPA3978 mutant phenotypes were suppressed by deleting , consistent with them having been caused by elevated MltF activity. We also discovered another interaction partner of PA3978 encoded by the PA5502 locus. The phenotypes of a ΔPA5502 mutant suggested that PA5502 interferes with the inhibitory function of PA3978 towards MltF, and we confirmed that activity for PA5502 . Therefore, PA3978 and PA5502 form an inhibitor/anti-inhibitor system that controls MltF activity. We propose to name these proteins Ilt (inhibitor of lytic transglycosylase) and Lii (lytic transglycosylase inhibitor, inhibitor).
IMPORTANCE
A peptidoglycan cell wall is an essential component of almost all bacterial cell envelopes, which determines cell shape and prevents osmotic rupture. Antibiotics that interfere with peptidoglycan synthesis have been one of the most important treatments for bacterial infections. Peptidoglycan must also be hydrolyzed to incorporate new material for cell growth and division, and to help accommodate important envelope-spanning systems. However, the enzymes that hydrolyze peptidoglycan must be carefully controlled to prevent autolysis. Exactly how this control is achieved is poorly understood in most cases, but is a highly active area of current research. Identifying hydrolase control mechanisms has the potential to provide new targets for therapeutic intervention. The work here reports the important discovery of a novel inhibitor/anti-nhibitor system that controls the activity of a cell wall hydrolase in the human pathogen , and which also affects resistance to an antibiotic used in the clinic.
PubMed: 37546783
DOI: 10.1101/2023.07.28.551027 -
Frontiers in Microbiology 2024Pholiota nameko is a widely consumed edible fungus. This study focuses on two crucial developmental stages of Pholiota nameko, namely, mycelium and ascospores. The...
INTRODUCTION
Pholiota nameko is a widely consumed edible fungus. This study focuses on two crucial developmental stages of Pholiota nameko, namely, mycelium and ascospores. The objectives of this research were to investigate changes in microbial diversity and community structure during the growth of Pholiota nameko and to analyze the adaptability of the dominant strains to their respective habitats through metabolic.
METHODS
Specifically, we conducted second-generation sequencing of the 16S rRNA gene (Illumina) on samples obtained from these stages. In addition, we isolated and characterized endophytes present in Pholiota nameko, focusing on examining the impact of dominant endophyte genera on autolysis. We also conducted a metabolic pathway analysis.
RESULTS AND DISCUSSION
The results unveiled 578,414 valid sequences of Pholiota nameko endophytic fungi. At the phylum level, the dominant taxa were Basidiomycota, Ascomycota, Zoopagomycota, and Mucoromycota. At the genus level, the dominant taxa observed were Pholiota, Inocybe, Fusarium, and Hortiboletus. For endophytic bacteria, we obtained 458,475 valid sequences. The dominant phyla were Proteobacteria, TM6, Firmicutes, and Bacteroidetes, while the dominant genera were Edaphobacter, Xanthomonas, Burkholderia, and Pseudomonas. Moreover, we identified the isolated strains in Pholiota nameko using 16S rDNA, and most of them were found to belong to the genus Pseudomonas, with Pseudomonas putida being the most prevalent strain. The findings revealed that the Pseudomonas putida strain has the ability to slow down the breakdown of soluble proteins and partially suppress the metabolic processes that generate superoxide anion radicals in Pholiota nameko, thereby reducing autolysis. Additionally, our results demonstrated that molybdenum enzyme-mediated anaerobic oxidative phosphorylation reactions were the primary energy metabolism pathway in the Pseudomonas putida strain. This suggests that the molybdenum cofactor synthesis pathway might be the main mechanism through which Pholiota nameko adapts to its complex and diverse habitats.
PubMed: 38690362
DOI: 10.3389/fmicb.2024.1319886 -
Journal of Animal Science Jan 2024Calpains are cysteine proteinases responsible for many biological roles in muscle, including protein degradation, muscle growth, and myoblast fusion. Calpains are...
Calpains are cysteine proteinases responsible for many biological roles in muscle, including protein degradation, muscle growth, and myoblast fusion. Calpains are inhibited by calpastatin, an endogenous inhibitor. Other factors, such as variations in pH, ionic strength, and oxidation influence calpain activity. This study aimed to determine the extent to which oxidation influences calpastatin inhibition of calpain-1. A series of order of addition assays were used to determine calpain-1 calcium activation and autolysis after exposure to an oxidizing agent (n-ethylmaleimide [NEM] or hydrogen peroxide [H2O2]. In the first series, purified calpastatin was added to the assay before or after oxidizing exposure at 165 mM NaCl, pH 6.5. In the second series, incubation buffer ionic strength (165 mM or 295 mM NaCl) was evaluated. The inhibitory activities of purified porcine calpastatin, purified human calpastatin domain I, or a subdomain B inhibitor peptide were evaluated in the third series. In the fourth series, a maleimide-polyethylene glycol molecule (MAL-PEG; MW = 5,000 Dalton) was used to evaluate the accessibility of free sulfhydryl groups and tagging of calpain-1 under each condition through a molecular weight shift assay. Results from this study indicate that autolysis of calpain-1, when used as an indicator of activation, occurred when the calpain-1/calpastatin complex was exposed to an oxidant or cysteine modifier such as NEM. However, when calpain-1 was exposed to the cysteine modifier before calpastatin, autolysis of calpain-1 did not occur or was significantly decreased (P < 0.05). Irreversible modification of cysteine residues by NEM prevented activation of calpain-1 in the absence of calpastatin, but if the cysteine modification is potentially reversible (H2O2), calpain-1 activity can be recovered. Results from this study indicate that when calpastatin is bound to calpain-1, calpain-1 activation can occur even after being exposed to a cysteine modifier (NEM) or hydrogen peroxide (H2O2). Calpain-1 is not tagged with maleimide-polyethylene glycol (MAL-PEG) in the presence of calpastatin, indicating that calpastatin blocks or covers free cysteines on calpain-1 from modification. Moreover, exposure to calpain-1/calpastatin complex with a cysteine modifier allows activation of calpain-1, indicating that the inhibitory action of calpastatin is compromised. These results indicate a regulatory role for calpastatin that is not inhibitory but protective for calpain-1.
Topics: Calpain; Calcium-Binding Proteins; Animals; Oxidation-Reduction; Hydrogen Peroxide; Swine; Calcium; Ethylmaleimide; Humans
PubMed: 38738874
DOI: 10.1093/jas/skae135 -
Infection and Drug Resistance 2024(MRSA) is a cause of staph infection that is difficult to treat because of resistance to some antibiotics. A recent study indicated that diarylurea is a novel...
BACKGROUND
(MRSA) is a cause of staph infection that is difficult to treat because of resistance to some antibiotics. A recent study indicated that diarylurea is a novel antibacterial agent against multi-drug resistant . In this work, we refined the bactericidal mechanism of as a peptidoglycan (PG) hydrolase by affecting AtlA-mediated PG homeostasis.
METHODS
A wild-type strain (WT) and a mutant strain (Δ) were used to investigate the effects of on the cell wall, PG, and autolysin regulatory system by antimicrobial susceptibility testing, hemolytic toxin assay, microanalysis, autolysis assay, qRT-PCR, ELISA and mouse model of pneumonia.
RESULTS
The results revealed that down-regulated the expression of genes related to peptidoglycan hydrolase (PGH) (, and ), and reduced the levels of PG, muramyl dipeptide (MDP), cytokines, and hemolytic toxin, while Δ interfered with the genes regulation and PG homeostasis. In the mouse MRSA pneumonia model, the same trend was observed in the nucleotide oligomerization domain protein 2 (NOD2) and relative proinflammatory factors.
CONCLUSION
may act as a novel inhibitor of PG hydrolyse, disrupting autolysin-mediated PG homeostasis, and reducing inflammation by down-regulating the MDP-NOD2 pathway.
PubMed: 38560705
DOI: 10.2147/IDR.S455339 -
Journal of Zoo and Wildlife Medicine :... Mar 2024Between 2015 and 2019, a health screening was carried out annually on captive-bred snails prior to export for reintroduction as part of an international effort to...
Between 2015 and 2019, a health screening was carried out annually on captive-bred snails prior to export for reintroduction as part of an international effort to repopulate areas of French Polynesia, where the snails were extinct or critically endangered. In total, 129 separate tank populations of 12 different species were screened at ZSL London Zoo. Wet mounts and smears stained with modified Ziehl-Neelsen (MZN) of 535 fecal samples were examined, and 45% contained flagellated protozoa, and 35.5% had MZN-positive oocysts, measuring 3-5 µm in diameter. Smaller (2 µm) presumptive spores, MZN-positive bacilli, ciliated protozoa and nematodes were recorded less frequently. Fecal bacterial culture yielded mixed species, with a clear predominance of species (88.9% of samples). The MZN-positive oocysts (3-5 µm) were present in 6.5% of impression smears from the apices of 432 snails examined postmortem, plus acid-fast bacilli in a few cases, but no 2 µm spores. Mixed bacteria were cultured from coelomic swabs, with species again the most common (63.5%). Histologic examination was carried out on 292 snails. Autolysis affected almost 90% of those found dead but only 3.4% of euthanized snails. Histology commonly identified microsporidial sporocysts in the digestive gland and midgut epithelium of all but two species. Intracellular, extracytoplasmic -like organisms were also common in the midgut but were only observed when snails were fixed in 10% formalin (2017-2019), not ethanol. There were no clear pathologic changes associated with either organism. Pigmented hemocytic nodules were commonly observed, most frequently in the foot process; these were either age related or evidence of prior chronic inflammatory reaction and of low clinical significance. With no evidence of poor health and no significant organisms found, a total of 4,978 individuals representing 12 species were exported for reintroduction.
Topics: Animals; Cryptosporidium; Cryptosporidiosis; Bacteria; Feces; Microsporidia
PubMed: 38453485
DOI: 10.1638/2023-0077