Correlation between bacterial extracellular vesicles and antibiotics: A potentially antibacterial strategy.

Bacterial extracellular vesicles (BEVs) are proteoliposome nanoparticles that are secreted by both Gram-negative (G-) and Gram-positive (G+) bacteria. BEVs have significant roles in various physiological processes of bacteria, including driving inflammatory responses, regulating bacterial pathogenesis, and promoting bacterial survival in diverse environments. Recently, there has been increasing interest in the use of BEVs as a potential solution to antibiotic resistance. BEVs have shown great promise as a new approach to antibiotics, as well as a drug-delivery tool in antimicrobial strategies. In this review, we provide a summary of recent scientific advances in BEVs and antibiotics, including BEV biogenesis, ability to kill bacteria, potential for delivering antibiotics, and their role in the development of vaccines or as immune adjuvants. We propose that BEVs provide a novel antimicrobial strategy that would be beneficial against the increasing threat of antibiotic resistance.

Baicalin Attenuates Panton-Valentine Leukocidin (PVL)-Induced Cytoskeleton Rearrangement via Regulating the RhoA/ROCK/LIMK and PI3K/AKT/GSK-3ß Pathways in Bovine Mammary Epithelial Cells.

Pore-forming toxins (PFTs) exert physiological effects by rearrangement of the host cell cytoskeleton. Staphylococcus aureus-secreted PFTs play an important role in bovine mastitis. In the study, we examined the effects of recombinant Panton-Valentine leukocidin (rPVL) on cytoskeleton rearrangement, and identified the signaling pathways involved in regulating the process in bovine mammary epithelial cells (BMECs) in vitro. Meanwhile, the underlying regulatory mechanism of baicalin for this process was investigated. The results showed that S. aureus induced cytoskeleton rearrangement in BMECs mainly through PVL. S. aureus and rPVL caused alterations in the cell morphology and layer integrity due to microfilament and microtubule rearrangement and focal contact inability. rPVL strongly induced the phosphorylation of cofilin at Ser3 mediating by the activation of the RhoA/ROCK/LIMK pathway, and resulted in the activation of loss of actin stress fibers, or the hyperphosphorylation of Tau at Ser396 inducing by the inhibition of the PI3K/AKT/GSK-3ß pathways, and decreased the microtubule assembly. Baicalin significantly attenuated rPVL-stimulated cytoskeleton rearrangement in BMECs. Baicalin inhibited cofilin phosphorylation or Tau hyperphosphorylation via regulating the activation of RhoA/ROCK/LIMK and PI3K/AKT/GSK-3ß signaling pathways. These findings provide new insights into the pathogenesis and potential treatment in S. aureus causing bovine mastitis.

The MADS-box transcription factor AoRlmA is involved in the regulation of mycelium development, conidiation, cell-wall integrity, stress response, and trap formation of Arthrobotrys oligospora.

The maintenance of cell-wall integrity (CWI) is important for mycelial growth, development, and pathogenicity in fungi. Arthrobotrys oligospora is a typical nematode-trapping (NT) fungus which can capture nematodes by producing adhesive networks. In this study, we characterized an orthologous MADS-box transcription factor RlmA (AoRlmA) downstream of the CWI regulatory pathway in A. oligospora. The deletion of AorlmA caused a reduction in mycelial growth, the number of nuclei, conidiation, and trap formation, as well as increased sensitivity to cell-wall synthesis-disrupting agents, osmotic agents, and oxidants; accordingly, the transcript levels of genes associated with sporulation, cell-wall biosynthesis, and DNA damage response were downregulated in the ΔAorlmA mutant. Furthermore, the absence of AorlmA resulted in a reduction in autophagy and endocytosis. Transcriptome analysis showed that differentially expressed genes in the absence of AorlmA were involved in membrane components, the oxidation-reduction process, transmembrane transport, metabolic processes, cellular components, organelles, cellular response to stress, and DNA damage response. In addition, metabolomic analysis showed that AoRlmA was involved in the regulation of secondary metabolites of A. oligospora. To summarize, our results highlighted the important roles of transcription factor RlmA in mycelial growth, conidiation, CWI, trap formation, stress response, autophagy, endocytosis, and secondary metabolism regulation in A. oligospora, providing a basis for elucidating the regulatory mechanism of the mycelial growth and development, pathogenicity, and stress response of NT fungi.

AoATG5 plays pleiotropic roles in vegetative growth, cell nucleus development, conidiation, and virulence in the nematode-trapping fungus Arthrobotrys oligospora.

Autophagy is an evolutionarily conserved process in eukaryotes, which is regulated by autophagy-related genes (ATGs). Arthrobotrys oligospora is a representative species of nematode-trapping (NT) fungi that can produce special traps for nematode predation. To elucidate the biological roles of autophagy in NT fungi, we characterized an orthologous Atg protein, AoAtg5, in A. oligospora. We found that AoATG5 deletion causes a significant reduction in vegetative growth and conidiation, and that the transcript levels of several sporulation-related genes were significantly downregulated during sporulation stage. In addition, the cell nuclei were significantly reduced in the ΔAoATG5 mutant, and the transcripts of several genes involved in DNA biosynthesis, repair, and ligation were significantly upregulated. In ΔAoATG5 mutants, the autophagic process was significantly impaired, and trap formation and nematocidal activity were significantly decreased. Comparative transcriptome analysis results showed that AoAtg5 is involved in the regulation of multiple cellular processes, such as autophagy, nitrogen metabolism, DNA biosynthesis and repair, and vesicular transport. In summary, our results suggest that AoAtg5 is essential for autophagy and significantly contributes to vegetative growth, cell nucleus development, sporulation, trap formation, and pathogenicity in A. oligospora, thus providing a basis for future studies focusing on related mechanisms of autophagy in NT fungi.

Functional analysis of seven regulators of G protein signaling (RGSs) in the nematode-trapping fungus Arthrobotrys oligospora.

Regulators of G protein signaling (RGSs) are proteins that negatively regulate G protein signal transduction. In this study, seven putative RGSs were characterized in the nematode-trapping (NT) fungus, Arthrobotrys oligospora. Deleting Rgs genes significantly increased intracellular cAMP levels, and caused defects in mycelia growth, stress resistance, conidiation, trap formation, and nematocidal activity. In particular, the ΔAoFlbA mutant was unable to produce conidia and traps. Transcriptomic analysis showed that amino acid metabolic and biosynthetic processes were significantly enriched in the ΔAoFlbA mutant compared to WT. Interestingly, Gas1 family genes are significantly expanded in A. oligospora and other NT fungi that produce adhesive traps, and are differentially expressed during trap formation in A. oligospora. Disruption of two Gas1 genes resulted in defective conidiation, trap formation, and pathogenicity. Our results indicate that RGSs play pleiotropic roles in regulating A. oligospora mycelial growth, development, and pathogenicity. Further, AoFlbA is a prominent member and required for conidiation and trap formation, possibly by regulating amino acid metabolism and biosynthesis. Our results provide a basis for elucidating the signaling mechanism of vegetative growth, lifestyle transition, and pathogenicity in NT fungi.

AoBck1 and AoMkk1 Are Necessary to Maintain Cell Wall Integrity, Vegetative Growth, Conidiation, Stress Resistance, and Pathogenicity in the Nematode-Trapping Fungus Arthrobotrys oligospora.

The cell wall integrity (CWI) pathway is composed of three mitogen-activated protein kinases (MAPKs), Bck1, Mkk1/2, and Slt2, and is one of the main signaling pathways for fungal pathogenesis, cell wall synthesis, and integrity maintenance. In this study, we characterized orthologs of Saccharomyces cerevisiae Bck1 and Mkk1 in the nematode-trapping (NT) fungus Arthrobotrys oligospora by multiple phenotypic comparison, and the regulation of conidiation and cell wall synthesis was analyzed using real-time PCR (RT-PCR). Both ΔAoBck1 and ΔAoMkk1 mutants showed severe defects in vegetative growth, cell nucleus number, and stress resistance. Both the mutants were unable to produce spores, and the transcription of several genes associated with sporulation and cell wall biosynthesis was markedly downregulated during the conidiation stage. Further, cell walls of the ΔAoBck1 and ΔAoMkk1 mutants were severely damaged, and the Woronin body failed to respond to cellular damage. In particular, the mutants lost the ability to produce mycelial traps for nematode predation. Taken together, AoBck1 and AoMkk1 play a conserved role in mycelial growth and development, CWI, conidiation, multi-stress tolerance, trap formation, and pathogenicity. We highlighted the role of AoBck1 and AoMkk1 in regulating the Woronin body response to cellular damage and cell nucleus development in A. oligospora.

Pleiotropic roles of Ras GTPases in the nematode-trapping fungus Arthrobotrys oligospora identified through multi-omics analyses.

The nematode-trapping fungi are ideal agents for controlling pathogenic nematodes. Arthrobotrys oligospora is a representative species of the same, producing traps for nematode predation. Here, three orthologous Ras GTPases (Ras2, Ras3, and Rheb) were characterized in A. oligospora. Our results indicate that they play pleiotropic roles in regulating the mycelial growth, conidiation, stress resistance, and pathogenicity of A. oligospora. Furthermore, deletion of Aoras2 and Aorheb significantly affected the mitochondrial activity, reactive oxygen species levels, lipid storage, and autophagy. Transcriptome analyses of ΔAoras2 mutant revealed that many repressed genes were associated with signal transduction, energy production, and carbohydrate transport and metabolism. Moreover, metabolic profile analyses showed that AoRas2 and AoRheb affect the biosynthesis of secondary metabolites in A. oligospora. Collectively, these findings provide an in-depth insight into the essential roles of Ras GTPases in vegetative growth, development, and pathogenicity and highlight their importance in the lifestyle switch of the nematode-trapping fungi.

Protein Kinase Ime2 Is Required for Mycelial Growth, Conidiation, Osmoregulation, and Pathogenicity in Nematode-Trapping Fungus Arthrobotrys oligospora.

Inducer of meiosis 2 (Ime2), a protein kinase that has been identified in diverse fungal species, functions in the regulation of various cellular processes, such as ascospore formation, pseudohyphal growth, and sexual reproduction. In this study, AoIme2, an ortholog of Saccharomyces cerevisiae Ime2, was characterized in the nematode-trapping fungus Arthrobotrys oligospora. Disruption of the gene Aoime2 caused defective growth, with slower mycelial growth in ΔAoime2 mutants than the wild type (WT) strain, and in the mutants, the number of hyphal septa in mycelia was higher and the number of cell nuclei in mycelia and conidia was considerably lower than in the WT strain. The conidial yields of the ΔAoime2 mutants were decreased by ∼33% relative to the WT strain, and the transcription of several sporulation-related genes, including abaA, fluG, rodA, aspB, velB, and vosA, was markedly downregulated during the conidiation stage. The ΔAoime2 mutants were highly sensitive to the osmotic stressors NaCl and sorbitol, and the cell wall of partial hyphae in the mutants was deformed. Further examination revealed that the cell wall of the traps produced by ΔAoime2 mutants became loose, and that the electron-dense bodies in trap cells were also few than in the WT strain. Moreover, Aoime2 disruption caused a reduction in trap formation and serine-protease production, and most hyphal traps produced by ΔAoime2 mutants did not form an intact hyphal loop; consequently, substantially fewer nematodes were captured by the mutants than by the WT strain. In summary, an Ime2-MAPK is identified here for the first time from a nematode-trapping fungus, and the kinase is shown to be involved in the regulation of mycelial growth and development, conidiation, osmolarity, and pathogenicity in A. oligospora.

Towards Al-induced manganese-containing superoxide dismutase inactivation and conformational changes: an integrating study with docking simulations.

Superoxide dismutase (SOD, EC 1.15.1.1) plays an important antioxidant defense role in skins exposed to oxygen. We studied the inhibitory effects of Al(3+) on the activity and conformation of manganese-containing SOD (Mn-SOD). Mn-SOD was significantly inactivated by Al(3+) in a dose-dependent manner. The kinetic studies showed that Al(3+) inactivated Mn-SOD follows the first-order reaction. Al(3+) increased the degree of secondary structure of Mn-SOD and also disrupted the tertiary structure of Mn-SOD, which directly resulted in enzyme inactivation. We further simulated the docking between Mn-SOD and Al(3+) (binding energy for Dock 6.3: -14.07 kcal/mol) and suggested that ASP152 and GLU157 residues were predicted to interact with Al(3+), which are not located in the Mn-contained active site. Our results provide insight into the inactivation of Mn-SOD during unfolding in the presence of Al(3+) and allow us to describe a ligand binding via inhibition kinetics combined with the computational prediction.

Trehalose has a protective effect on human brain-type creatine kinase during thermal denaturation.

We investigated the effects of trehalose on thermal inactivation and aggregation of human brain-type creatine kinase (hBBCK) in this study. In the presence of 1.0 M trehalose, the midpoint temperature of thermal inactivation (T (m)) of hBBCK increased by 4.6 °C, and the activation energy (E (a)) for thermal inactivation increased from 29.7 to 41.1 kJ mol(-1). Intrinsic fluorescence spectra also showed an increase in the apparent transition temperature (T (1/2)) of hBBCK from 43.0 °C to 46.5 °C, 47.7 °C, and 49.9 °C in 0, 0.6, 0.8, and 1.2 M trehalose, respectively. In addition, trehalose significantly blocked the aggregation of hBBCK during thermal denaturation. Our results indicate that trehalose has potential applications as a thermal stabilizer and may aid in the folding of other enzymes in addition to hBBCK.