Discovery of Salifungin as a Repurposed Antibiotic against Methicillin-Resistant Staphylococcus aureus with Limited Resistance Development.

Exploring novel antimicrobial drugs and strategies has become essential to the fight MRSA-associated infections. Herein, we found that membrane-disrupted repurposed antibiotic salifungin had excellent bactericidal activity against MRSA, with limited development of drug resistance. Furthermore, adding salifungin effectively decreased the minimum inhibitory concentrations of clinical antibiotics against Staphylococcus aureus. Evaluations of the mechanism demonstrated that salifungin disrupted the level of H+ and K+ ions using hydrophilic and lipophilic groups to interact with bacterial membranes, causing the disruption of bacterial proton motive force followed by impacting on bacterial the function of the respiratory chain and adenosine 5'-triphosphate, thereby inhibiting phosphatidic acid biosynthesis. Moreover, salifungin also significantly inhibited the formation of bacterial biofilms and eliminated established bacterial biofilms by interfering with bacterial membrane potential and inhibiting biofilm-associated gene expression, which was even better than clinical antibiotics. Finally, salifungin exhibited efficacy comparable to or even better than that of vancomycin in the MRSA-infected animal models. In conclusion, these results indicate that salifungin can be a potential drug for treating MRSA-associated infections.

Altered metabolome and microbiome associated with compromised intestinal barrier induced hepatic lipid metabolic disorder in mice after subacute and subchronic ozone exposure.

Exposure to ozone has been associated with metabolic disorders in humans, but the underlying mechanism remains unclear. In this study, the role of the gut-liver axis and the potential mechanism behind the metabolic disorder were investigated by histological examination, microbiome and metabolome approaches in mice during the subacute (4-week) and subchronic (12-week) exposure to 0.5 ppm and 2.5 ppm ozone. Ozone exposure resulted in slowed weight gain and reduced hepatic lipid contents in a dose-dependent manner. After exposure to ozone, the number of intestinal goblet cells decreased, while the number of tuft cells increased. Tight junction protein zonula occludens-1 (ZO-1) was significantly downregulated, and the apoptosis of epithelial cells increased with compensatory proliferation, indicating a compromised chemical and physical layer of the intestinal barrier. The hepatic and cecal metabolic profiles were altered, primarily related to lipid metabolism and oxidative stress. The abundance of Muribaculaceae increased dose-dependently in both colon and cecum, and was associated with the decrease of metabolites such as bile acids, betaine, and L-carnitine, which subsequently disrupted the intestinal barrier and lipid metabolism. Overall, this study found that subacute and subchronic exposure to ozone induced metabolic disorder via disturbing the gut-liver axis, especially the intestinal barrier. These findings provide new mechanistic understanding of the health risks associated with environmental ozone exposure and other oxidative stressors.

EvfG is a multi-function protein located in the Type VI secretion system for ExPEC.

The Type VI secretion system (T6SS) functions as a protein transport nanoweapon in several stages of bacterial life. Even though bacterial competition is the primary function of T6SS, different bacteria exhibit significant variations. Particularly in Extraintestinal pathogenic Escherichia coli (ExPEC), research into T6SS remains relatively limited. This study identified the uncharacterized gene evfG within the T6SS cluster of ExPEC RS218. Through our experiments, we showed that evfG is involved in T6SS expression in ExPEC RS218. We also found evfG can modulate T6SS activity by competitively binding to c-di-GMP, leading to a reduction in the inhibitory effect. Furthermore, we found that evfG can recruit sodA to alleviate oxidative stress. The research shown evfG controls an array of traits, both directly and indirectly, through transcriptome and additional tests. These traits include cell adhesion, invasion, motility, drug resistance, and pathogenicity of microorganisms. Overall, we contend that evfG serves as a multi-functional regulator for the T6SS and several crucial activities. This forms the basis for the advancement of T6SS function research, as well as new opportunities for vaccine and medication development.

Antibacterial efficacy and mechanism of Cyprinus carpio chemokine-derived L-10 against multidrug-resistant Escherichia coli infections.

OBJECTIVES: Antimicrobial resistance has raised concerns regarding untreatable infections and poses a growing threat to public health. Rational design of new AMPs is an ideal solution to this threat. METHODS: In this study, we designed, modified, and synthesised an excellent AMP, L-10, based on the original sequence of the Cyprinus carpio chemokine. All experimental data were presented as the mean ± standard deviation (SD), and the two-tailed unpaired T-test method was used to analyze all data. RESULTS: L-10 exhibited excellent antibacterial activity with negligible toxicity and improved the efficacy of a broad class of antibiotics against MDR Gram-negative pathogens, including tetracycline, meropenem, levofloxacin, and rifampin. Mechanistic studies have suggested that L-10 targets the bacterial membrane components, LPS and PG, to disrupt bacterial membrane integrity, thereby exerting antibacterial effects and enhancing the efficacy of antibiotics. Moreover, in animal infection models, L-10 significantly increased the survival rate of infected animals and effectively reduced the tissue bacterial load and inflammatory factor levels. In addition to its direct antibacterial activity, L-10 dramatically reduced pulmonary pathological alterations in a mouse model of endotoxemia and suppressed LPS-induced proinflammatory cytokines in vitro and in vivo. Lastly, L-10 was successfully expressed in Pichia pastoris and maintained antimicrobial activity against MDR Gram-negative pathogens in vivo and in vitro. CONCLUSION: Collectively, these results reveal the potential of L-10 as an ideal candidate against MDR bacterial infections and provide new insights into the design, development, and clinical application of AMPs.

Repurposing an Antioxidant to Kill Mycobacterium tuberculosis by Targeting the 50S Subunit of the Ribosome.

Tuberculosis and drug-resistant TB remain serious threats to global public health. It is urgent to develop novel anti-TB drugs in order to control it. In addition to redesigning and developing new anti-TB drugs, drug repurposing is also an innovative way to develop antibacterial drugs. Based on this method, we discovered SKQ-1 in the FDA-approved drug library and evaluated its anti-TB activity. In vitro, we demonstrated that SKQ-1 engaged in bactericidal activity against drug-sensitive and -resistant Mtb and confirmed the synergistic effects of SKQ1 with RIF and INH. Moreover, SKQ-1 showed a significant Mtb-killing effect in macrophages. In vivo, both the SKQ-1 treatment alone and the treatment in combination with RIF were able to significantly reduce the bacterial load and improve the survival rate of G. mellonella infected with Mtb. We performed whole-genome sequencing on screened SKQ-1-resistant strains and found that the SNP sites were concentrated in the 50S ribosomal subunit of Mtb. Furthermore, we proved that SKQ-1 can inhibit protein translation. In summary, from the perspective of drug repurposing, we discovered and determined the anti-tuberculosis effect of SKQ-1, revealed its synergistic effects with RIF and INH, and demonstrated its mechanism of action through targeting ribosomes and disrupting protein synthesis, thus making it a potential treatment option for DR-TB.

Impact of Airborne Pathogen-Derived Extracellular Vesicles on Macrophages Revealed by Raman Spectroscopy and Multiomics.

Long-term exposure to the indoor environment may pose threats to human health due to the presence of pathogenic bacteria and their byproducts. Nanoscale extracellular vesicles (EVs) extensively secreted from pathogenic bacteria can traverse biological barriers and affect physio-pathological processes. However, the potential health impact of EVs from indoor dust and the underlying mechanisms remain largely unexplored. Here, Raman spectroscopy combined with multiomics (genomics and proteomics) was used to address these issues. Genomic analysis revealed that Pseudomonas was an efficient producer of EVs that harbored 68 types of virulence factor-encoding genes. Upon exposing macrophages to environmentally relevant doses of Pseudomonas aeruginosa PAO1-derived EVs, macrophage internalization was observed, and release of inflammatory factors was determined by RT-PCR. Subsequent Raman spectroscopy and unsupervised surprisal analysis of EV-affected macrophages distinguished metabolic alterations, particularly in proteins and lipids. Proteomic analysis further revealed differential expression of proteins in inflammatory and metabolism-related pathways, indicating that EV exposure induced macrophage metabolic reprogramming and inflammation. Collectively, our findings revealed that pathogen-derived EVs in the indoor environments can act as a new mediator for pathogens to exert adverse health effects. Our method of Raman integrated with multiomics offers a complementary approach for rapid and in-depth understanding of EVs' impact.

First report of sweet corn root rot caused by Pythium graminicola in southeast China.

Sweet corn (Zea mays L.) is widely consumed as fresh or frozen vegetable worldwide, and Zhengtian68 is a popular commercial variety cultivated extensively in southeast China. In May 2021, 40% of the inbred line YK063 (the female parent of Zhengtian68) showed early yellowing of the leaves at flowering time in a commercial seed production field with a total area of 0.5 ha in Guangzhou, Guangdong Province after a heavy rain. Black and rotten roots were observed in the diseased plants after digging the whole plant out of the soil. Grain filling was also severely affected, adversely impacting seed production. Diseased plants were more easily found in the lower section of the field, where water accumulated after rainfall. Three plants with rotten roots were collected randomly from the field to identify the causal pathogen. The diseased roots were cut into 2-3 mm sections, washed in 75% ethanol for 2 minutes and rinsed three times in sterile distilled water. Four to five sections per plant were placed on potato dextrose agar (PDA) and incubated at 28℃ in the dark for three days. Three isolates GF1, GF2, and GF3 from different plants were purified by hyphal tip isolation and transferred to new PDA and 10% V8 juice agar (16 g agar, 3 g CaCO3, 100 ml V8 juice, and 900 ml distilled water) and incubated at 28℃ for 10 days in darkness for further investigation. Translucent, glassy mycelial growth was observed on the PDA media. Morphological characteristics of the 3 isolates were observed under a microscope from the 10%V8 media. The hyphae were aseptate and 2.7 to 4.5 µm wide (mean±SD,3.3±0.44µm, n=44). Sporangia were inflated, or lobulate, terminal, or intercalary. Oogonia were globose, smooth-walled, terminal, or occasionally intercalary, with a diameter of 17.2-24.1 µm (mean±SD, 21.3±2.14µm, n=29). Oospores were globose, plerotic, smooth, and 14.5-21.2 µm (mean±SD, 18.7±2.07µm, n=35) in diameter. The antheridia were diclinous or monoclinous, not intercalary, and one to six antheridia were attached to each oogonium. Based on these morphological characteristics, 3 isolates were identified as Pythium spp. including Pythium graminicola (Van der Plaats-Niterink 1981). Genomic DNA was extracted from the mycelia grown on PDA using a Fungal Genomic DNA kit (Scintol, Beijing, China) according to the manufacturer's instructions. The cytochrome oxidase II (Cox II) gene and internal transcribed spacer (ITS) region of the rDNA were amplified using the primers FM58/FM66 (Martin 2000) and ITS4/ITS5 (White et al. 1990) respectively. Amplification was performed in a 50µl reaction volume using 25 µl PCR Mix (Trans Gene, Beijing, China), 3 µl genomic DNA (50 ng/µl), 1 µl each forward and reverse primer (10 µM), and 20 µl ddH2O. The PCR program was as follows: initial denaturation at 95°C for 30 s, 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 60 s, extension at 72°C for 60 s, and a final extension at 72°C for 10 min. PCR products were sequenced and submitted to GenBank (accession no. OQ504322, OQ933130, and OQ933212 for ITS; OQ512002, OQ942203, and OQ942204 for Cox II). BLASTn analysis revealed that the ITS and Cox II sequences showed more than 98.62% similarity (721/724bp, 722/724bp,723/724bp for ITS; 514/514bp, 506/507bp, 500/507bp for Cox II) to P. graminicola ATCC96234 (accession no. AB095045 for ITS, and AB160849 for Cox II), respectively, supporting the morphological analysis. A neighbor-joining phylogenetic analysis of the ITS and Cox II concatenated sequence further confirmed that the isolates were P. graminicola. To test the pathogenicity of GF1, GF2, and GF3 a wheat seed inoculum was prepared as previously described (Qu et al. 2016). Sweet corn YK063 plants were planted in sterilized nutrient soil in plastic pots (one plant per pot) and grown in a greenhouse at 28℃ with 60% humidity and a 12-h/12-h light-dark cycle. For each isolate,10 plants were inoculated with 20 infected wheat seeds around the roots at the V5 stage, while 10 other YK063 plants were inoculated with the non-infected wheat seeds as a control. The experiment was repeated once. Three weeks later, the non-inoculated plants were asymptomatic. In contrast, inoculated plants showed stunning, yellowing of the leaves, root rot, and decreased production of lateral roots, exhibiting symptoms similar to those originally described for the disease. P. graminicola was successfully reisolated from the diseased roots and identified by morphological characteristics and sequencing of the ITS and Cox II as the causal agent for this root rot disease, fulfilling Koch's postulate for defining a causal agent. P. graminicola was reported as a causal agent of damping-off on dent corn in Georgia (Li et al. 2018). To our knowledge, this is the first report of P. graminicola causing root rot in sweet corn in southeast China. Identification of this pathogen will facilitate further research on this disease and the development of effective strategies to control the disease.

Microbial modifications with Lycium barbarum L. oligosaccharides decrease hepatic fibrosis and mitochondrial abnormalities in mice.

BACKGROUND: Lycium barbarum L. is a typical Chinese herbal and edible plant and are now consumed globally. Low molecular weight L. barbarum L. oligosaccharides (LBO) exhibit better antioxidant activity and gastrointestinal digestibility in vitro than high molecular weight polysaccharides. However, the LBO on the treatment of liver disease is not studied. PURPOSE: Modification of the gut microbial ecosystem by LBO is a promising treatment for liver fibrosis. STUDY DESIGN AND METHODS: Herein, LBO were prepared and characterized. CCl4-treated mice were orally gavaged with LBO and the effects on hepatic fibrosis and mitochondrial abnormalities were evaluated according to relevant indicators (gut microbiota, faecal metabolites, and physiological and biochemical indices). RESULTS: The results revealed that LBO, a potential prebiotic source, is a pyranose cyclic oligosaccharide possessing α-glycosidic and ß-glycosidic bonds. Moreover, LBO supplementation restored the configuration of the bacterial community, enhanced the proliferation of beneficial species in the gastrointestinal tract (e.g., Bacillus, Tyzzerella, Fournierella and Coriobacteriaceae UCG-002), improved microbial metabolic alterations (i.e., carbohydrate metabolism, vitamin metabolism and entero-hepatic circulation), and increased antioxidants, including doxepin, in mice. Finally, LBO administration reduced serum inflammatory cytokine and hepatic hydroxyproline levels, improved intestinal and hepatic mitochondrial functions, and ameliorated mouse liver fibrosis. CONCLUSION: These findings indicate that LBO can be utilized as a prebiotic and has a remarkable ability to mitigate liver fibrosis.

Canagliflozin Inhibited the Activity of Hemolysin and Reduced the Inflammatory Response Caused by Streptococcus suis.

Highly virulent Streptococcus suis (S. suis) infections can cause Streptococcal toxic shock-like syndrome (STSLS) in pigs and humans, in which an excessive inflammatory response causes severe damage. Hemolysin (SLY) is a major virulence factor of S. suis serotype 2 that produces pores in the target cell membrane, leading to cytoplasmic K+ efflux and activation of the NLRP3 inflammasome, ultimately causing STSLS. The critical aspect of hemolysin in the pathogenesis of S. suis type 2 makes it an attractive target for the development of innovative anti-virulence drugs. Here, we use the S. suis toxin protein (SLY) as a target for virtual screening. A compound called canagliflozin, a hypoglycemic agent, was identified through screening. Canagliflozin significantly inhibits the hemolytic activity of hemolysin. The results combined with molecular dynamics simulation, surface plasmon resonance, and nano differential scanning fluorimetry show that canagliflozin inhibits the hemolytic activity of SLY by binding to SLY. In addition, canagliflozin markedly reduced the release of SC19-induced inflammatory factors at the cellular level and in mice. Importantly, the combination of canagliflozin and ampicillin had a 90% success rate in mice, significantly greater than the therapeutic effect of ampicillin. The findings suggest that canagliflozin may be a promising new drug candidate for S. suis infections.

Gut microbiota related response of Oryzias melastigma to combined exposure of polystyrene microplastics and tetracycline.

The co-existence of microplastics (MPs) and antibiotics in the coastal environment poses a combined ecological risk. Single toxic effects of MPs or antibiotics on aquatic organisms have been verified, however, the exploration of their combined toxic effects remains limited. Here, foodborne polystyrene microplastics (PS-MPs, 10 µm, 0.1 % w/w in food) and waterborne tetracyclines (TC, 50 µg/L) were used to expose an estuarine fish Oryzias melastigma for four weeks. We found that the aqueous availability of TC was not significantly altered coexisting with MPs. The fish body weight gain was significantly slower in TC alone or combined groups than the control group, consistent with the lower lipid content in livers. The body length gain was significantly inhibited by the combined presence compared to the single exposure. Both exposures led to a shift of gut microbiota composition and diversity. TC and the combined group possessed similar gut microbiota which is distinct from PS-MPs and the control group. The Firmicutes/Bacteroidetes (F/B) ratio in the TC and combined groups were significantly lower compared to the control, while the PS-MPs group showed no significant impact. Metabolomic analysis of the fish liver confirmed the shift of metabolites in specific pathways after different exposures. More, a number of gut microbiota-related metabolites on lipid metabolism was perturbed, which were annotated in arachidonic acid metabolism and linoleic acid metabolism. In all, TC modulates bacterial composition in the fish gut and disturbs their liver metabolites via the gut-liver axis, which led to the slower growth of O. melastigma. More, the adverse impact was aggravated by the co-exposure to foodborne PS-MPs.

Effectors of the Type VI Secretion System Have the Potential to Be Modified into Antimicrobial Peptides.

The use of antibiotics has led to the emergence of multidrug-resistant (MDR) bacteria, and there is an urgent need to find alternative treatments to alleviate this pressure. The type VI secretion system (T6SS) is a protein delivery system present in bacterial cells that secretes effectors that participate in bacterial virulence. Given the potential for the transformation of these effectors into antimicrobial peptides (AMPs), we designed T6SS effectors into AMPs that have a membrane-disrupting effect. These effectors kill bacteria by altering the membrane potential and increasing the intracellular reactive oxygen species (ROS) content. Moreover, AMPs also have a significant therapeutic effect both in vivo and in vitro. This finding suggests that it is possible to modify bacterial components of bacteria themselves to create compounds that fight bacteria. IMPORTANCE This study first identified and modified the T6SS effector into positively charged alpha-helical peptides. These peptides have good antibacterial and bactericidal effects on G+ bacteria and G- bacteria. This study broadens the source of AMPs and makes T6SS effectors more useful.

Analysis of the noncoding RNA regulatory networks of H37Rv- and H37Rv△1759c-infected macrophages.

Noncoding RNAs regulate the process of Mycobacterium tuberculosis (M. tb) infecting the host, but there is no simultaneous transcriptional information of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) and the global regulatory networks of non-coding RNA. Rv1759c, a virulence factor, is a member of protein family containing the proline-glutamic acid (PE) in M. tb, which can increase M. tb survival. To reveal the noncoding RNA regulatory networks and the effect of Rv1759c on non-coding RNA expression during M. tb infection, we collected samples of H37Rv- and H37Rv△1759c-infected macrophages and explored the full transcriptome expression profile. We found 356 mRNAs, 433 lncRNAs, 168 circRNAs, and 12 miRNAs differentially expressed during H37Rv infection, 356 mRNAs, 433 lncRNAs, 168 circRNAs, and 12 miRNAs differentially expressed during H37Rv△1759c infection. We constructed lncRNA/circRNA-miRNA-mRNA regulatory networks during H37Rv and H37Rv△1759c infection. We demonstrated the role of one of the hubs of the networks, hsa-miR-181b-3p, for H37Rv survival in macrophages. We discovered that the expression changes of 68 mRNAs, 92 lncRNAs, 26 circRNAs, and 3 miRNAs were only related to the deletion of Rv1759c by comparing the transcription profiles of H37Rv and H37Rv△1759c. Here, our study comprehensively characterizes the transcriptional profiles in THP1-derived-macrophages infected with H37Rv and H37Rv△1759c, which provides support and new directions for in-depth exploration of noncoding RNA and PE/PPE family functions during the infection process.

First report of bacterial leaf streak in sweet corn caused by Pantoea ananatis in southeast China.

Sweet corn (Zea mays convar. saccharata var. rugosa) is a popular vegetable crop in southeast China. During the spring seasons of 2018-2021, a serious outbreak of bacterial leaf streak was observed in sweet corn variety Yuetian28 in the field in Guangzhou, Guangdong Province. The disease incidence was 50%-70%. Infected leaves initially displayed long, chlorotic streaks parallel to veins at the V5-V6 stage, and then turned white or brown and dried out over the course of disease development. In severe infections, leaf lesion coalesced to form large irregular blight areas (Fig. S1A). To investigate this disease, we collected 0.5 cm2 samples of infected leaves from four plants after surface sterilization and rinsed them three times with sterile distilled water. We placed all leaf samples on nutrient agar (NA) medium and incubated them at 28℃ for 48 hours. Bright-yellowish colonies were observed near the edges of the samples. We picked the colonies and re-streaked them onto NA medium three times to obtain pure cultures. Four isolates, GZ2201, GZ2202, GZ2203, and GZ2204, were selected for further study. All isolates were gram-negative rods and were negative for oxidase, urease, nitrate reductase reactions, and gelatin liquefaction. They were positive for catalase, citrate utilization, indole production, and the Voges-Proskauer test. We sequenced the 16S rDNA, rpoB, leuS, and gyrB sequences using previously reported primers (Brady et al. 2008) and deposited the sequences in GenBank (accession nos. ON740665 to ON740668 for 16S rDNA; ON755167 to ON755170 for rpoB; ON755171 to ON755174 for leuS; and OP227136 to OP227139 for gyrB). The sequences share >98% identity with sequences from Pantoea ananatis type strain LMG2665 (GenBank JFZU01) indicating that the causal pathogen of bacteria leaf streak of sweet corn is P. ananatis (Fig. S1B). Phylogenetic analysis of gyrB, leuS, and rpoB concatenated sequence showed that the four isolates clustered with P. ananatis (Fig S2). To test the pathogenicity of the isolates of P. ananatis on the sweet corn variety Yuetian28, we inoculated plants at the V3 stage by syringe infiltration of bacterial suspension (108 CFU/ml) (Kini et al. 2020) or sterile distilled water as a negative control. Inoculated plants were placed in a growth chamber at 28 ℃, 60% relative humidity, 16-h/8-h light-dark cycle. After 7 days of incubation, chlorotic streaks resembling the original symptoms developed on inoculated plants (Fig. S1D), while control plants remained symptomless (Fig. S1C). We successfully re-isolated bacteria from the inoculated plants and confirmed their identity by sequencing of 16S rDNA, rpoB, leuS, and gyrB. P. ananatis was previously reported to cause leaf spot disease in maize grown in Argentina, Ecuador, and China (Alippi et al. 2010; Toaza et al. 2021; Cui et al. 2022). To our knowledge, this is the first report of P. ananatis causing leaf streak in sweet corn in southeast China. Further research on P. ananatis management is needed to help control disease spread.

Identification of an anti-virulence drug that reverses antibiotic resistance in multidrug resistant bacteria.

The persistent incidence of high levels of multidrug-resistant (MDR) bacteria seriously endangers global public health. In response to MDR-associated infections, new antibacterial drugs and strategies are particularly needed. Screening to evaluate a potential compound to reverse antibiotic resistance is a good strategy to alleviate this crisis. In this paper, using high-throughput screening methods, we identified that oxyclozanide potentiated tetracycline antibiotics act against MDR bacterial pathogens by promoting intracellular accumulation of tetracycline in resistant bacteria. Furthermore, mechanistic studies demonstrated that oxyclozanide could directly kill bacteria by disrupting bacterial membrane and inducing the overproduction of bacterial reactive oxygen species. Oxyclozanide effectively reduced the production of virulence proteins in S. aureus and neutralized the produced α-hemolysin, thereby effectively alleviating the inflammatory response caused by bacteria. Finally, oxyclozanide significantly reversed tetracycline resistance in animal infection assays. In summary, these results demonstrated the capacity of oxyclozanide as a novel antibiotic adjuvant, antibacterial and anti-virulence multifunctional compound to circumvent MDR bacteria and improve the therapeutic effect of persistent infections caused by MDR bacteria worldwide.

MiR-25 blunts autophagy and promotes the survival of Mycobacterium tuberculosis by regulating NPC1.

Mycobacterium tuberculosis (Mtb) evades host clearance by inhibiting autophagy. MicroRNA-25 (miR-25) expression was significantly up-regulated in the lung tissues of mice infected with Bacillus Calmette-Guerin (BCG) and macrophages infected with Mtb or BCG, especially in the early stages of infection. MiR-25 can significantly increase the survival of Mtb and BCG in macrophages. We validated that miR-25 targets the NPC1 protein located on the lysosomal membrane, resulting in damage to lysosomal function, thereby inhibiting autophagolysosome formation and promoting the survival of Mtb and BCG. Consistently, mice lacking miR-25 exhibited more resistant to BCG infection. In addition, we found that Rv1759c induces the expression of miR-25 through NFKB inhibitor zeta (NFKBIZ). This study demonstrates that the role of miR-25 during Mtb infection contributes to a better understanding of the pathogenesis of tuberculosis (TB).

Integrated metabolomic and transcriptomic analysis identifies benzo[a]pyrene-induced characteristic metabolic reprogramming during accumulation of lipids and reactive oxygen species in macrophages.

Polycyclic aromatic hydrocarbon exposure is a major risk factor for cardiovascular diseases. Macrophage lipid accumulation is a characteristic molecular event in the pathophysiology of cardiovascular diseases. Metabolic reprogramming is an intervention target for diseases and toxic effects of environmental pollutants. However, comprehensive metabolic reprogramming related to BaP-induced macrophage lipid accumulation is currently unexplored. Therefore, metabolomics and transcriptomics were conducted to unveil relevant metabolic reprogramming in BaP-exposed macrophages, and to discover potential intervention targets. Metabolomics revealed that most amino acids, nucleotides, monosaccharides, and organic acids were significantly decreased, while most fatty acids and steroids accumulated in BaP-exposed macrophages. Transcriptomics showed that fatty acid synthesis and oxidation, and steroid synthesis and export were decreased, while import of fatty acids and steroids was increased, indicating potential roles of lipid transport in macrophage lipid accumulation following BaP exposure. Meanwhile, alanine, aspartate and glutamate metabolism, branched-chain amino acid degradation, nucleotide synthesis, monosaccharide import, pentose phosphate pathway, citrate synthesis, and glycolysis were decreased, while nucleotide degradation was increased, thus inducing decreases in most amino acids, nucleotides, monosaccharides, and organic acids in BaP-exposed macrophages. Additionally, increases in oxidative stress and the activation of antioxidant systems were observed in BaP-exposed macrophages, which was evinced by increases in reactive oxygen species, and the activation of Fenton reaction, Vdac2/3, Sod2, and Nrf2. Moreover, BaP-induced accumulation of reactive oxygen species and lipids in macrophages could be abolished by epigallocatechin-3-gallate. Quantitative PCR showed that BaP exposure activated aryl hydrocarbon receptor signaling and promoted the proinflammatory phenotype in macrophages, and these effects were inhibited or even abolished by the separate treatment with epigallocatechin-3-gallate or CH-223191, suggesting the regulatory role of aryl hydrocarbon receptor signaling in BaP-induced toxic effects. This study provides novel insights into the toxic effects of polycyclic aromatic hydrocarbons on macrophage metabolism and potential intervention targets.

5-HT2A Receptor and 5-HT Degradation Play a Crucial Role in Atherosclerosis by Modulating Macrophage Foam Cell Formation, Vascular Endothelial Cell Inflammation, and Hepatic Steatosis.

AIM: Previously, we found that diabetes-related liver dysfunction is due to activation of the 5-HT2A receptor (5-HT2AR) and increased synthesis and degradation of 5-HT. Here, we investigated the role of 5-HT in the development of atherosclerosis. METHODS: The study was conducted using high-fat diet-fed male ApoE-/- mice, THP-1 cell-derived macrophages, and HUVECs. Protein expression and biochemical indexes were determined by Western blotting and quantitative analysis kit, respectively. The following staining methods were used: oil red O staining (showing atherosclerotic plaques and intracellular lipid droplets), immunohistochemistry (showing the expression of 5-HT2AR, 5-HT synthase, and CD68 in the aortic wall), and fluorescent probe staining (showing intracellular ROS). RESULTS: In addition to improving hepatic steatosis, insulin resistance, and dyslipidemia, co-treatment with a 5-HT synthesis inhibitor and a 5-HT2AR antagonist significantly suppressed the formation of atherosclerotic plaques and macrophage infiltration in the aorta of ApoE-/- mice in a synergistic manner. Macrophages and HUVECs exposed to oxLDL or palmitic acid in vitro showed that activated 5-HT2AR regulated TG synthesis and oxLDL uptake by activating PKCε, resulting in formation of lipid droplets and even foam cells; ROS production was due to the increase of both intracellular 5-HT synthesis and mitochondrial MAO-A-catalyzed 5-HT degradation, which leads to the activation of NF-κB and the release of the inflammatory cytokines TNF-α and IL-1ß from macrophages and HUVECs as well as MCP-1 release from HUVECs. CONCLUSION: Similar to hepatic steatosis, the pathogenesis of lipid-induced atherosclerosis is associated with activation of intracellular 5-HT2AR, 5-HT synthesis, and 5-HT degradation.

miR-495 Regulates Cellular Reactive Oxygen Species Levels by Targeting sod2 To Inhibit Intracellular Survival of Mycobacterium tuberculosis in Macrophages.

Mycobacterium tuberculosis is a chronic infectious disease pathogen. To date, tuberculosis is a major infectious disease that endangers human health. To better prevent and treat tuberculosis, it is important to study the pathogenesis of M. tuberculosis. Based on early-stage laboratory research results, in this study, we verified the upregulation of sod2 in Bacillus Calmette-Guérin (BCG) and H37Rv infection. By detecting BCG/H37Rv intracellular survival in sod2-silenced and sod2-overexpressing macrophages, sod2 was found to promote the intracellular survival of BCG/H37Rv. miR-495 then was determined to be downregulated by BCG/H37Rv. BCG/H37Rv can upregulate sod2 expression by miR-495 to promote the intracellular survival of BCG/H37Rv through a decline in ROS levels. This study provides a theoretical basis for developing new drug targets and treating tuberculosis.

General characteristics of the influence of surfactants on the bacteriolytic activity of lysozyme based on the example of enzymatic lysis of Lactobacillus plantarum cells in the presence of Tween 21 and SDS.

The general characteristics of the effect of surfactants on the activity of lysozyme were demonstrated. The kinetics of bacterial cell lysis is consistent with the Michaelis-Menten equation and the presence of surfactants does not shift the pH-optimum of activity. Surfactants do not change the Km value but instead, affect the Vmax value. The experimental dependencies are well described by theoretical equations, which assume three surfactant binding sites on the lysozyme molecule. The dependencies of the activity of lysozyme on the surfactant concentration are either a step type (i.e., a higher plateau becomes a lower plateau), or a dependency with a maximum and continuation of the curve in the form of a plateau but with an increase in the surfactant concentration. It can be assumed that there is a mechanism for the regulation of lysozyme activity by an unknown natural factor that has a suitable hydrophobic radical capable of binding to the surface of lysozyme.

Function of Rhs proteins in porcine extraintestinal pathogenic Escherichia coli PCN033.

Extraintestinal pathogenic Escherichia coli (ExPEC) is an important zoonotic pathogen that places severe burdens on public health and animal husbandry. There are many pathogenic factors in E. coli. The type VI secretion system (T6SS) is a nano-microbial weapon that can assemble quickly and inject toxic effectors into recipient cells when danger is encountered. T6SSs are encoded in the genomes of approximately 25% of sequenced Gram-negative bacteria. When these bacteria come into contact with eukaryotic cells or prokaryotic microbes, the T6SS assembles and secretes associated effectors. In the porcine ExPEC strain PCN033, we identified four classic rearrangement hotspot (Rhs) genes. We determined the functions of the four Rhs proteins through mutant construction and protein expression. Animal infection experiments showed that the Δrhs-1CT, Δrhs-2CT, Δrhs-3CT, and Δrhs-4CT caused a significant decrease in the multiplication ability of PCN033 in vivo. Cell infection experiments showed that the Rhs protein is involved in anti-phagocytosis activities and bacterial adhesion and invasion abilities. The results of this study demonstrated that rhs1, rhs3, and rh4 plays an important role in the interaction between PCN033 and host cell. Rhs2 has contribution to cell and mice infection. This study helps to elucidate the pathogenic mechanism governing PCN033 and may help to establish a foundation for further research seeking to identify potential T6SS effectors.