MiR-184 targeting FOXO1 regulates host-cell oxidative stress induced by Chlamydia psittaci via the Wnt/ß-catenin signaling pathway.

Chlamydia psittaci is a human pathogen that causes atypical pneumonia after zoonotic transmission. We confirmed that C. psittaci infection induces oxidative stress in human bronchial epithelial (HBEs) cells and explored how this is regulated through miR-184 and the Wnt/ß-catenin signaling pathway. miR-184 mimic, miR-184 inhibitor, FOXO1 siRNA, or negative control sequence was transfected into HBE cells cultured in serum-free medium using Lipofectamine 2000. Then, prior to the cells were infected with C. psittaci 6BC, and the cells were treated with or without 30 µM Wnt/ß-catenin inhibitor ICG-001. Quantification of reactive oxygen species, malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione was carried out according to the manufacturer's protocol using a corresponding assay kit. The outcome of both protein and gene was measured by western blotting or real-time fluorescence quantitative PCR. In C. psittaci-infected HBE cells, miR-184 was upregulated, while one of its target genes, FOXO1, was downregulated. ROS and MDA levels increased, while SOD and GSH contents decreased after C. psittaci infection. When miR-184 expression was downregulated, the level of oxidative stress caused by C. psittaci infection was reduced, and the Wnt/ß-catenin signaling pathway was inhibited. The opposite results were seen when miR-184 mimic was used. Transfecting with FOXO1 siRNA reversed the effect of miR-184 inhibitor. Moreover, when the Wnt/ß-catenin-specific inhibitor ICG-001 was used, the level of oxidative stress induced by C. psittaci infection was significantly suppressed. miR-184 can target FOXO1 to promote oxidative stress in HBE cells following C. psittaci infection by activation of the Wnt/ß-catenin signaling pathway.

Pyroptosis in microbial infectious diseases.

Pyroptosis is a gasdermins-mediated programmed cell death that plays an essential role in immune regulation, and its role in autoimmune disease and cancer has been studied extensively. Increasing evidence shows that various microbial infections can lead to pyroptosis, associated with the occurrence and development of microbial infectious diseases. This study reviews the recent advances in pyroptosis in microbial infection, including bacterial, viral, and fungal infections. We also explore potential therapeutic strategies for treating microbial infection-related diseases by targeting pyroptosis.

Chlamydia psittaci Induces Autophagy in Human Bronchial Epithelial Cells via PERK and IRE1α, but Not ATF6 Pathway.

Chlamydia psittaci is an important pathogen that causes chronic and atypical pneumonia in humans. Autophagy and the unfolded protein response (UPR) are important mechanisms for regulating the growth of infectious parasitic pathogens in living cells. Here, we explored whether C. psittaci infection induced autophagy via the UPR and the effect of these cellular responses on the survival and replication of C. psittaci in human bronchial epithelial cells (HBEs). Not only were the numbers of autophagosomes and the expression of LC3-II and Beclin1 increased following C. psittaci infection of HBEs, but also the expression of p62 (also called sequestosome-1) was downregulated. Moreover, after C. psittaci infection, the UPR and UPR sensors PERK/eIF2α and IRE1α/XBP1 were activated, but not the ATF6 pathway. When either Bip siRNA was used to block normal initiation of the UPR, or activation of the PERK and IER1α pathways was blocked with specific inhibitors GSK2606414 and 4µ8C, the level of autophagy caused by C. psittaci infection was significantly inhibited. Furthermore, blocking activation of the UPR and associated pathways significantly reduced the number of C. psittaci inclusions. Our research suggests that the UPR, via the PERK and IRE1α, but not ATF6 signaling pathways, regulates HBE-cell autophagy induced by C. psittaci infection and the replication of C. psittaci.

Fluorescent Biosensor Based on Hairpin DNA Stabilized Copper Nanoclusters for Chlamydia trachomatis Detection.

Chlamydia trachomatis (C. trachomatis) is a kind of intracellular parasitic microorganism, which can causes many diseases such as trachoma. In this strategy, a specific hairpin DNA with the probe loop as specific regions to recognize C. trachomatis DNA with strong affinity was designed, and its stem consisted of 24 AT base pairs as an effective template for hairpin DNA-CuNCs formation. In the absence of C. trachomatis DNA, the detection system showed strong orange fluorescence emission peaks at 606 nm. In the presence of C. trachomatis DNA, the conformation of DNA probe changed after hybridizing with C. trachomatis DNA. Then, the amount of hairpin DNA-CuNCs was reduced and resulted in low fluorescence emission. C. trachomatis DNA displayed a significant inhibitory effect on the synthesis of fluorescent hairpin DNA-CuNCs due to the competition between C. trachomatis DNA and the specific hairpin DNA. Under the optimal experimental conditions, different concentrations of C. trachomatis were tested and the results showed a good linear relationship in the range of 50 nM to 950 nM. Moreover, the detection limit was 18.5 nM and this detection method possessed good selectivity. Finally, the fluorescent biosensor had been successfully applied to the detection of C. trachomatis target sequence in HeLa cell lysate, providing a new strategy for the detection of C. trachomatis.

Analysis of microRNA expression profiles in human bronchial epithelial cells infected by Chlamydia psittaci.

BACKGROUND: Chlamydia psittaci is a pathogen of birds that can cause zoonotic disease in mammals including pneumonia in humans. MicroRNAs (miRNAs) are a class of small non-coding RNA fragments with a length of about 22 nt, which play an important role in regulating gene expression after transcription. Chlamydia infection can cause changes in host cell miRNA expression, but the potential biological function of miRNAs in C. psittaci infection and pathogenesis is not well understood. METHODS: Small RNA sequencing (sRNA-Seq) technology was used to characterise miRNA expression in human bronchial epithelial (HBE) cells after C. psittaci infection, and differentially expressed miRNAs were identified. Candidate target genes for these miRNAs were then functionally annotated by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The sRNA-Seq results were partially validated by quantitative real time polymerase chain reaction (qRT-PCR) and miRNA-target networks were constructed using visualization software. RESULTS: We identified 151 differentially expressed miRNAs (46 known miRNAs and 105 novel miRNAs) in C. psittaci-infected HBE cells, of which 140 were upregulated and 11 were downregulated. Of these, 17 known miRNAs were significantly upregulated and two were downregulated using P < 0.05 and |log2FoldChange|>1.5 as threshold criteria. GO enrichment results showed that the predicted targets of these differentially expressed miRNAs were mainly involved in transcriptional regulation and ATP binding. KEGG pathway analysis suggested that the candidate target genes were involved in several important signaling pathways such as MAPK, ErbB, cGMP-PKG, cAMP, mTOR, GNRH, oxytocin, PI3K-Akt and AMPK, which are primarily related to biological processes such as transcription and signal transduction. The qRT-PCR results for miR-2116-3p, miR-3195, miR-663a, miR-10401-5p, miR-124-3p, miR-184, miR-744-5p and hsa-miR-514b-5p were consistent with the sRNA-Seq data. CONCLUSIONS: A large amount of miRNA expression profile data relating to C. psittaci infection was obtained, which provides a useful experimental and theoretical basis for further understanding the pathogenic mechanisms of C. psittaci infection.

Isolation and genomic analysis of temperate phage 5W targeting multidrug-resistant Acinetobacter baumannii.

Temperate phages are potential therapeutic agents, but only a few temperate phages infecting multidrug-resistant Acinetobacter baumannii have been identified. In this study, we isolated 5W, a temperate phage that infects multidrug-resistant A. baumannii, from pond water using the enrichment method. A member of the Siphoviridae family, 5W has a narrow host range and infected only four of 19 A. baumannii clinical isolates. It exhibited rapid adsorption (> 90% in 6 min), a latency period of 20 min, and a burst size of ~ 180 plaque-forming units (PFU/cell). 5W contains a linear double-stranded DNA (dsDNA) genome of 43,032 bp with a GC content of 39.85%. The 5W genome contains 61 open reading frames, including lysogen-forming genes, but lacks any known virulence and antibiotic resistance genes. The lysin of 5W is an N-acetyl-ß-D-muramidase belonging to the GH_108 family. The α-helical structure and highly positively charged amino acids in the C-terminal region indicate potential antibacterial activity against A. baumannii, and the M/S subunits of the restriction endonuclease are inserted into the lysogenic gene cluster. Comparative genome analysis revealed high similarity with two different prophages in A. baumannii ABCR01, suggesting that 5W may be derived from recombination of other prophages.

The roles of microRNAs played in lung diseases via regulating cell apoptosis.

MicroRNAs (miRNAs) are a type of endogenous non-coding short-chain RNA, which plays a crucial role in the regulation of many essential cellular functions, including cellular migration, proliferation, invasion, autophagy, oxidative stress, apoptosis, and differentiation. The lung can be damaged by pathogenic microorganisms, as well as physical or chemical factors. Research has confirmed that miRNAs and lung cell apoptosis can affect the development and progression of several lung diseases. This article reviews the role of miRNAs in the development of lung disease through regulating host cell apoptosis.

Recent advances on aptamer-based biosensors for detection of pathogenic bacteria.

As a significant constituent in biosphere, bacteria have a great influence on human activity. The detection of pathogen bacteria is closely related to the human health. However, the traditional methods for detection of pathogenic bacteria are time-consuming and difficult for quantification, although they are practical and reliable. Therefore, novel strategies for rapid, sensitive, and cost-effective detection are in great demand. Aptamer is a kind of oligonucleotide that selected by repeated screening in vitro or systematic evolution of ligands by exponential enrichment (SELEX) technology. Over the past years, owing to high affinity and specificity of aptamers, a variety of aptamer-based biosensors have been designed and applied for pathogen detection. In this review, we have discussed the recent advances on the applications of aptamer-based biosensors in detection of pathogenic bacteria. In addition, we also point out some problems in current methods and look forward to the further development of aptamer-based biosensors for pathogen detection.

Deficiency of LIGHT signaling pathway exacerbates Chlamydia psittaci respiratory tract infection in mice.

LIGHT, a costimulatory member of the immunoglobulin superfamily (Ig SF), can greatly impact T cell activation. The role of the LIGHT signaling pathway in chlamydial infection was evaluated in mice following respiratory tract infection with Chlamydia psittaci. Compared with wild type (WT) mice, LIGHT knockout (KO) mice showed significant reduction of body weight, much lower survival rate, higher bacterial burden, prolonged infection time courses and more severe pathological changes in lung tissue. The mRNA levels of IFN-γ, TNF-α, IL-17 and IL-12 in the lung tissue of LIGHT KO mice were significantly lower than those in WT mice. While there was no obvious difference in the percentages of CD4+ and CD8+ T cells in the spleens of the two groups of mice, there was a markedly elevated percentage of CD4+ CD25+ FoxP3+ Treg cells in LIGHT KO mice. Together, these results demonstrate that the LIGHT signaling pathway is not only required for inflammatory cytokine production as part of the host response to chlamydial infection, but also influences the differentiation of CD4+ CD25+ FoxP3+ Treg cells, both of which may be essential for control of C. psittaci respiratory tract infection.

[Isolation and characterization of siphovirus phages infecting bovine Streptococcus agalactiae].

OBJECTIVE: To isolate and identify Streptococcus agalactiae phages and screen candidate phages to control infection caused by bovine S. agalactiae. METHODS: We used two methods for isolation of S. agalactiae phages, namely (1) isolation of phages from milk and environmental samples, and (2) isolation of phages via induction of lysogens with Mitomycin C. Double-layer agar culture method was used to purify phages. Then the newly obtained phages, with S. agalactiae phage JX01 isolated from mastitis milk, were comparatively analyzed in the following aspects: morphology of phages by transmission electron microscopy, host range of phages to 55 S. agalactiae strains and other Streptococcus strains, phages DNA using EcoR I, Xba I, Pst I and Sal I, the optical multiplicity of infection, absorption curve and one step growth curve, and the stability of phages at different storage conditions. RESULTS: The comparative analysis of the 3 novel phages LYGO9, HZ04 and pA11 (induced from S. agalctiae bovine clinical isolate HAJL2011070601) with JX01 showed that the 4 phages were classified as the member of Siphovirdae family. EcoR I, Sal I, Xba I and Pst I separately digested the 4 phages DNA provided 4, 3, 3 and 2 profiles, respectively. This suggested that they were different strains. All the 4 phages specifically infected bovine S. agalactiae isolates. LYGO9, pA11, JX01 and HZ04 could lyse 12, 13, 20 and 23 of 42 tested bovine S. agalctiae isolates, respectively. This clearly indicated that these 4 phages are closely related. CONCLUSION: The 3 new phages which specifically lyse bovine S. agalactiae isolates are siphovirus phages. Phage LYGO9 was shown having a short latent period and a larger burst size.

Characterization and complete genome sequence analysis of Staphylococcus aureus bacteriophage JS01.

Staphylococcus aureus is a primary pathogen that causes bovine mastitis resulting in serious economic losses and herd management problems in dairy cows. A novel bacteriophage, JS01, specifically infecting bovine S. aureus, was isolated from milk of mastitis-affected cattle. TEM observation showed that it belonged to the family Siphovirus. The JS01 strain demonstrated a broad host range. The prediction result of PHACTS suggested that the JS01 strain was temperate phage. The JS01 genome is 43,458 bp long, with a GC content of 33.32% and no tRNAs. Annotation and functional analysis of the predicted ORFs revealed six functional groups: structure and morphology, DNA replication and regulation, packaging, lysogeny, lysis, and pathogenicity. Comparative analysis between JS01, S. aureus MSSA476, and S. aureus prophage PVL was also performed. The characterization and genomic analysis of JS01 provide a better understanding of S. aureus-targeting bacteriophages and useful information for the development of phage-based biocontrol agents against S. aureus.

Target-triggered dual signal amplification based on HCR-enhanced nanozyme activity for the sensitive visual detection of Escherichia coli.

The detection of foodborne pathogens is crucial for food hygiene regulation and disease diagnosis. Colorimetry has become one of the main analytical methods in studying foodborne pathogens due to its advantages of visualization, low cost, simple operation, and no complex instrument. However, the low sensitivity limits its applications in early identification and on-site detection for trace analytes. In order to overcome such a limitation, herein we propose a joint strategy featuring dual signal amplification based on the hybridization chain reaction (HCR) and DNA-enhanced peroxidase-like activity of gold nanoparticles (AuNPs) for the sensitive visual detection of Escherichia coli. Target bacteria bound specifically to the aptamer domain in the capture hairpin probe, exposing the trigger domain for HCR and forming the extended double-stranded DNA (dsDNA) structures. The peroxidase-like catalytic capacity of AuNPs can be enhanced significantly by dsDNAs with the sticky ends of dsDNAs being adsorbed on AuNPs and the rigidity of dsDNAs causing the spatial regulation of AuNP concentration. The intensity of the enhancement was linearly related to the number of target bacteria. With the above strategy, the detection limit of our colorimetric method for Escherichia coli was down to 28 CFU mL-1 within a short analytical time (50 min). This study provides a new perspective for the sensitive and visual detection of early bacterial contamination in foods.

Characterization and genome sequencing of a novel bacteriophage infecting Streptococcus agalactiae with high similarity to a phage from Streptococcus pyogenes.

A novel bacteriophage, JX01, specifically infecting bovine Streptococcus agalactiae was isolated from milk of mastitis-affected cattle. The phage morphology showed that JX01 belongs to the family Siphoviridae, and this phage demonstrated a broad host range. Microbiological characterization demonstrated that nearly 90 % of JX01 phage particles were adsorbed after 2.5 min of incubation, that the burst size was 20 virions released per infected host cell, and that there was a latent period of 30 min. JX01 was thermal sensitive and showed acid and alkaline resistance (pH 3-11). The genome of JX01 was found to consist of a linear, double-stranded 43,028-bp DNA molecule with a GC content of 36.81 % and 70 putative open reading frames (ORFs) plus one tRNA. Comparative genome analysis revealed high similarity between JX01 and the prophage 315.2 of Streptococcus pyogenes.

Enhanced Peroxidase-Like and Antibacterial Activity of Ir-CoatedPd-Pt Nanodendrites as Nanozyme.

To inhibit the growth of bacteria, the DA-PPI nanozyme with enhanced peroxidase-like activity was synthesized. The DA-PPI nanozyme was obtained by depositing high-affinity element iridium (Ir) on the surface of Pd-Pt dendritic structures. The morphology and composition of DA-PPI nanozyme were characterized using SEM, TEM, and XPS. The kinetic results showed that the DA-PPI nanozyme possessed a higher peroxidase-like activity than that of Pd-Pt dendritic structures. The PL, ESR, and DFT were employed to explain the high peroxidase activity. As a proof of concept, the DA-PPI nanozyme could effectively inhibit E. coli (G-) and S. aureus (G+) due to its high peroxidase-like activity. The study provides a new idea for the design of high active nanozymes and their application in the field of antibacterial.

The regulation roles of miRNAs in Helicobacter pylori infection.

Helicobacter pylori is a kind of Gram-negative bacteria that parasitizes on human gastric mucosa. Helicobacter pylori infection is very common in human beings, which often causes gastrointestinal diseases, including chronic gastritis, duodenal ulcer and gastric cancer. MicroRNAs are a group of endogenous non-coding single stranded RNAs, which play an important role in cell proliferation, differentiation, autophagy, apoptosis and inflammation. In recent years, relevant studies have found that the expression of microRNA is changed after Helicobacter pylori infection, and then regulate the biological process of host cells. This paper reviews the regulation role of microRNAs on cell biological behavior through different signal pathways after Helicobacter pylori infection.

Neuroprotection by Nrf2 via modulating microglial phenotype and phagocytosis after intracerebral hemorrhage.

Activated microglia are divided into pro-inflammatory and anti-inflammatory functional states. In anti-inflammatory state, activated microglia contribute to phagocytosis, neural repair and anti-inflammation. Nrf2 as a major endogenous regulator in hematoma clearance after intracerebral hemorrhage (ICH) has received much attention. This study aims to investigate the mechanism underlying Nrf2-mediated regulation of microglial phenotype and phagocytosis in hematoma clearance after ICH. In vitro experiments, BV-2 cells were assigned to normal group and administration group (Nrf2-siRNA, Nrf2 agonists Monascin and Xuezhikang). In vivo experiments, mice were divided into 5 groups: sham, ICH + vehicle, ICH + Nrf2-/-, ICH + Monascin and ICH + Xuezhikang. In vitro and in vivo, 72 h after administration of Monascin and Xuezhikang, the expression of Nrf2, inflammatory-associated factors such as Trem1, TNF-α and CD80, anti-inflammatory, neural repair and phagocytic associated factors such as Trem2, CD206 and BDNF were analyzed by the Western blot method. In vitro, fluorescent latex beads or erythrocytes were uptaken by BV-2 cells in order to study microglial phagocytic ability. In vivo, hemoglobin levels reflect the hematoma volume. In this study, Nrf2 agonists (Monascin and Xuezhikang) upregulated the expression of Trem2, CD206 and BDNF while decreased the expression of Trem1, TNF-α and CD80 both in vivo and in vitro. At the same time, after Monascin and Xuezhikang treatment, the phagocytic capacity of microglia increased in vitro, neurological deficits improved and hematoma volume lessened in vivo. These results were reversed in the Nrf2-siRNA or the Nrf2-/- mice. All these results indicated that Nrf2 enhanced hematoma clearance and neural repair, improved neurological outcomes through enhancing microglial phagocytosis and alleviating neuroinflammation.

Pathogenetic characterization of a Micrococcus luteus strain isolated from an infant.

Purpose: To explore the clinical characteristics of Micrococcus luteus bloodstream infection in an infant and characterize the phenotype and genotype of the isolated strains, as well as seek suitable infection models for assessing virulence. Methods: Clinical data was collected from an infant patient diagnosed with M. luteus bloodstream infection. Metagenomic sequencing was performed on the isolated blood sample. The strain was isolated and underwent mass spectrometry, biochemical tests, antibiotic susceptibility assays, and whole-genome sequencing. The Galleria mellonella infection model was used to assess M. luteus virulence. Results: Patient responded poorly to cephalosporins, but recovered after Linezolid treatment. Metagenomic sequencing identified M. luteus as the predominant species in the sample, confirming infection. They were identified as M. luteus with a high confidence level of 98.99% using mass spectrometry. The strain showed positive results for Catalase, Oxidase, and Urea tests, and negative results for Mannose, Xylose, Lactose, Mannitol, Arginine, and Galactose tests, consistent with the biochemical profile of M. luteus reference standards. M. luteus susceptibility to 15 antibiotics was demonstrated and no resistance genes were detected. Predicted virulence genes, including clpB, were associated with metabolic pathways and the type VI secretion system. The infection model demonstrated dose-dependent survival rates. Conclusion: The infant likely developed a bloodstream infection with M. luteus due to compromised immunity. Although the isolated strain is sensitive to cephalosporin antibiotics and has low pathogenicity in infection models, clinical treatment with cephalosporins was ineffective. Linezolid proved to be effective, providing valuable guidance for future clinical management of such rare infections.

Distribution of drug-resistant genes in alveolar lavage fluid from patients with psittacosis and traceability analysis of causative organisms.

Background: Chlamydia psittaci is a small bacterium often found in birds, including poultry, and domesticated mammals, which causes psittacosis (or parrot fever) in humans. Different strains of C. psittaci respond variably to antibiotics, suggesting a possible risk of antibiotic resistance. In general, different genotypes of C. psittaci have relatively stable hosts and different pathogenicity. Methods: Macrogenomic sequencing was performed using nucleic acids extracted from psittacosis patients' alveolar lavage fluid samples and analyzed for genetic variability and antibiotic resistance genes. Nucleic acid amplification sequences specific to the core coding region of the C. psittaci ompA gene were used, and a phylogenetic tree was constructed with C. psittaci genotypic sequences from other sources, including Chinese published sources. The C. psittaci found in each patient were genotyped by comparing ompA gene sequences. In addition, to better illustrate the relationship between genotype and host of C. psittaci, 60 bird fecal samples were collected from bird-selling stores for screening and C. psittaci typing. Results: Macrogenomic sequence alignment revealed the presence of resistance genes in varying abundance in samples from all three patients, including C. psittaci resistance gene sequences from two patients that matched those previously published on NCBI. Based on ompA genotyping, two patients were infected with C. psittaci genotype A and one patient was infected with genotype B. All five C. psittaci-positive samples obtained from bird-selling stores were genotype A. Both genotypes are reported to be infectious to humans. The host origin of the samples and the previously reported main sources of each genotype suggested that all but one of the C. psittaci genotype A in this study were derived from parrots, while genotype B was probably derived from chickens. Conclusion: The presence of bacterial resistance genes in psittacosis patients may affect the efficacy of clinical antibiotic therapy. Focusing on the developmental progression of bacterial resistance genes and differences in the therapeutic efficacy may facilitate effective treatment of clinical bacterial infections. Pathogenicity genotypes (e.g., genotype A and genotype B) are not limited to one animal host, suggesting that monitoring the development and changes of C. psittaci may help prevent transmission to humans.

Mechanisms of Oxidative Stress and Therapeutic Targets following Intracerebral Hemorrhage.

Oxidative stress (OS) is induced by the accumulation of reactive oxygen species (ROS) following intracerebral hemorrhage (ICH) and plays an important role in secondary brain injury caused by the inflammatory response, apoptosis, autophagy, and blood-brain barrier (BBB) disruption. This review summarizes the current state of knowledge regarding the pathogenic mechanisms of brain injury after ICH, markers for detecting OS, and therapeutic strategies that target OS to mitigate brain injury.

Microglia: A Double-Edged Sword in Intracerebral Hemorrhage From Basic Mechanisms to Clinical Research.

Microglia are the resident immune cells of the central nervous system (CNS). It is well established that microglia are activated and polarized to acquire different inflammatory phenotypes, either pro-inflammatory or anti-inflammatory phenotypes, which act as a critical component in the neuroinflammation following intracerebral hemorrhage (ICH). Microglia produce pro-inflammatory mediators at the early stages after ICH onset, anti-inflammatory microglia with neuroprotective effects appear to be suppressed. Previous research found that driving microglia towards an anti-inflammatory phenotype could restrict inflammation and engulf cellular debris. The principal objective of this review is to analyze the phenotypes and dynamic profiles of microglia as well as their shift in functional response following ICH. The results may further the understanding of the body's self-regulatory functions involving microglia following ICH. On this basis, suggestions for future clinical development and research are provided.