Enolase of Streptococcus suis serotype 2 promotes biomolecular condensation of ribosomal protein SA for HBMECs apoptosis.

BACKGROUND: Ribosomal protein SA (RPSA) of human brain microvascular endothelial cells (HBMECs) can transfer from the cytosol to the cell surface and act as a receptor for some pathogens, including Streptococcus suis serotype 2 (SS2), a zoonotic pathogen causing meningitis in pigs and humans. We previously reported that SS2 virulence factor enolase (ENO) binds to RPSA on the cell surface of HBMECs and induces apoptosis. However, the mechanism that activates RPSA translocation to the cell surface and induces ENO-mediated HBMEC apoptosis is unclear. RESULTS: Here, we show that RPSA localization and condensation on the host cell surface depend on its internally disordered region (IDR). ENO binds to the IDR of RPSA and promotes its interaction with RPSA and vimentin (VIM), which is significantly suppressed after 1,6-Hexanediol (1,6-Hex, a widely used tool to disrupt phase separation) treatment, indicating that ENO incorporation and thus the concentration of RPSA/VIM complexes via co-condensation. Furthermore, increasing intracellular calcium ions (Ca2+) in response to SS2 infection further facilitates the liquid-like condensation of RPSA and aggravates ENO-induced HBMEC cell apoptosis. CONCLUSIONS: Together, our study provides a previously underappreciated molecular mechanism illuminating that ENO-induced RPSA condensation activates the migration of RPSA to the bacterial cell surface and stimulates SS2-infected HBMEC death and, potentially, disease progression. This study offers a fresh avenue for investigation into the mechanism by which other harmful bacteria infect hosts via cell surfaces' RPSA.

Rapid and facile detection of largemouth bass ranavirus with CRISPR/Cas13a.

Largemouth bass ranavirus (LMBV) is an epidemic disease that seriously jeopardizes the culture of largemouth bass（Micropterus salmoides）, and it has a very high incidence in largemouth bass. Once an outbreak occurs, it may directly lead to the failure of the culture, resulting in substantial economic losses, but there is no effective vaccine or special effective drug yet. Consequently, it is important to establish an accurate, sensitive, convenient and specific detection approach for preventing LMBV infection. The recombinant enzyme-assisted amplification (RAA) technology was used in combination with clustered regularly interspaced short palindromic repeats (CRISPR), and associated protein 13a (CRISPR/Cas13a) to detect LMBV. We designed RAA primers and CRISPR RNA (crRNA) that targeted the conserved region in the LMBV main capsid protein (MCP) gene, amplified sample nucleic acids using the RAA technology, performed CRISPR/Cas13a fluorescence detection and evaluated the sensitivity and specificity of the established method with qPCR as a control method. This technique was able to determine the results by collecting fluorescence signals, visualizing fluorescence by UV excitation and combining with lateral flow strips (LFS). The sensitivity and specificity of the established method were consistent with the qPCR method. Besides, it was performed at a constant temperature of 37 °C and the sensitivity of the reaction system was 3.1 × 101 copies/µL, with no cross-reactivity with other common aquatic pathogens. Further, the positive detection rate of the proposed method in 32 clinical samples was consistent with that of qPCR. In conclusion, our established RAA-CRISPR/Cas13 method for detecting LMBV is sensitive, simple and specific, which is applicable in the rapid on-site detection and epidemiological monitoring of LMBV.

High-dimensional analysis reveals an immune atlas and novel neutrophil clusters in the lungs of model animals with Actinobacillus pleuropneumoniae-induced pneumonia.

Due to the increase in bacterial resistance, improving the anti-infectious immunity of the host is rapidly becoming a new strategy for the prevention and treatment of bacterial pneumonia. However, the specific lung immune responses and key immune cell subsets involved in bacterial infection are obscure. Actinobacillus pleuropneumoniae (APP) can cause porcine pleuropneumonia, a highly contagious respiratory disease that has caused severe economic losses in the swine industry. Here, using high-dimensional mass cytometry, the major immune cell repertoire in the lungs of mice with APP infection was profiled. Various phenotypically distinct neutrophil subsets and Ly-6C+ inflammatory monocytes/macrophages accumulated post-infection. Moreover, a linear differentiation trajectory from inactivated to activated to apoptotic neutrophils corresponded with the stages of uninfected, onset, and recovery of APP infection. CD14+ neutrophils, which mainly increased in number during the recovery stage of infection, were revealed to have a stronger ability to produce cytokines, especially IL-10 and IL-21, than their CD14- counterparts. Importantly, MHC-II+ neutrophils with antigen-presenting cell features were identified, and their numbers increased in the lung after APP infection. Similar results were further confirmed in the lungs of piglets infected with APP and Klebsiella pneumoniae infection by using a single-cell RNA-seq technique. Additionally, a correlation analysis between cluster composition and the infection process yielded a dynamic and temporally associated immune landscape where key immune clusters, including previously unrecognized ones, marked various stages of infection. Thus, these results reveal the characteristics of key neutrophil clusters and provide a detailed understanding of the immune response to bacterial pneumonia.

Untargeted metabolomics reveals alternations in metabolism of bovine mammary epithelial cells upon IFN-γ treatment.

BACKGROUND: IFN-γ is a pleiotropic cytokine that has been shown to affect multiple cellular functions of bovine mammary epithelial cells (BMECs) including impaired milk fat synthesis and induction of malignant transformation via depletion of arginine, one of host conditionally essential amino acids. But the molecular mechanisms of these IFN-γ induced phenotypes are still unknown. METHODS: BMECs were treated with IFN-γ for 6 h, 12 h, and 24 h. The metabolomic profiling in BMECs upon IFN-γ induction were assessed using untargeted ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) metabolomic analysis. Key differentially expressed metabolites (DEMs) were quantified by targeted metabolomics. RESULTS: IFN-γ induction resulted in significant differences in the contents of metabolites. Untargeted analysis identified 221 significantly DEMs, most of which are lipids and lipid-like molecules, organic acids and derivatives, organ heterocyclic compounds and benzenoids. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, DEMs were enriched in fructose and mannose metabolism, phosphotransferase system (PTS), ß-alanine metabolism, arginine and proline metabolism, methane metabolism, phenylalanine metabolism, and glycolysis/gluconeogenesis. Quantification of selected key DEMs by targeted metabolomics showed significantly decreased levels of D-(-)-mannitol, argininosuccinate, and phenylacetylglycine (PAG), while increased levels in S-hydroxymethylglutathione (S-HMG) and 2,3-bisphospho-D-glyceric acid (2,3-BPG). CONCLUSIONS: These results provide insights into the metabolic alterations in BMECs upon IFN-γ induction and indicate potential theoretical basis for clarifying IFN-γ-induced diseases in mammary gland.

Potential of phage EF-N13 as an alternative treatment strategy for mastitis infections caused by multidrug-resistant Enterococcus faecalis.

Bovine mastitis is the most common and costly disease affecting dairy cattle throughout the world. Enterococcus faecalis is one of the environmental origin mastitis-causing pathogens. The treatment of bovine mastitis is primarily based on antibiotics. Due to the negative impact of developing antibiotic resistance and adverse effects on soil and water environments, the trend toward use of nonantibiotic treatments is increasing. Phages may represent a promising alternative treatment strategy. However, it is unknown whether phages have therapeutic effects on E. faecalis-induced mastitis. Thus, the objective of this study was to investigate the degree of protection conferred by a phage during murine mastitis caused by multidrug-resistant E. faecalis. Enterococcus faecalis was isolated from the milk of dairy cows with mastitis, and a phage was isolated using the E. faecalis isolates as hosts. The bactericidal ability of the phage against E. faecalis and the ability to prevent biofilm formation were determined in vitro. The therapeutic potential of the phage on murine mastitis was evaluated in vivo. We isolated 14 strains of E. faecalis from the milk of cows with mastitis, all of which exhibited multidrug resistance, and most (10/14) could form strong biofilms. Subsequently, a new phage (EF-N13) was isolated using the multidrug-resistant E. faecalis N13 (isolated from mastitic milk) as the host. The phage EF-N13 belongs to the family Myoviridae, which has short latent periods (5 min) and high bursts (284 pfu/cell). The genome of EF-N13 lacked bacterial virulence-, antibiotic resistance-, and lysogenesis-related genes. Furthermore, bacterial loading in the raw milk medium was significantly reduced by EF-N13 and was unaffected by potential IgG antibodies. In fact, EF-N13 could effectively prevent the formation of biofilm by multidrug-resistant E. faecalis. All of these characteristics suggest that EF-N13 has potential as mastitis therapy. In vivo, 1 × 105 cfu/gland of multidrug-resistant E. faecalis N13 resulted in mastitis development within 24 h. A single dose of phage EF-N13 (1 × 104, 1 × 105, or 1 × 106 pfu/gland) could significantly decrease bacterial counts in the mammary gland at 24 h postinfection. Histopathological observations demonstrated that treatment with phage EF-N13 effectively alleviated mammary gland inflammation and damage. This effect was confirmed by the lower levels of proinflammatory cytokines IL-6, IL-1ß, and tumor necrosis factor-α in the mammary gland treated with phage EF-N13 compared with those treated with phosphate-buffered saline. Overall, the data underscored the potential of phage EF-N13 as an alternative therapy for bovine mastitis caused by multidrug-resistant E. faecalis.

LAP3 contributes to IFN-γ-induced arginine depletion and malignant transformation of bovine mammary epithelial cells.

BACKGROUND: IFN-γ has been traditionally recognized as an inflammatory cytokine that involves in inflammation and autoimmune diseases. Previously we have shown that sustained IFN-γ induced malignant transformation of bovine mammary epithelial cells (BMECs) via arginine depletion. However, the molecular mechanism underlying this is still unknown. METHODS: In this study, the amino acids contents in BMECs were quantified by a targeted metabolomics method. The acquisition of differentially expressed genes was mined from RNA-seq dataset and analyzed bioinformatically. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting, and immunohistochemistry (IHC) assay were performed to detect gene mRNA and protein expression levels. CCK-8 and would healing assays were used to detect cell proliferation and migration abilities, respectively. Cell cycle phase alternations were analyzed by flow cytometry. RESULTS: The targeted metabolomics analysis specifically discovered IFN-γ induced arginine depletion through accelerating arginine catabolism and inhibiting arginine anabolism in BMECs. Transcriptome analysis identified leucine aminopeptidase 3 (LAP3), which was regulated by p38 and ERK MAPKs, to downregulate arginine level through interfering with argininosuccinate synthetase (ASS1) as IFN-γ stimulated. Moreover, LAP3 also contributed to IFN-γ-induced malignant transformation of BMECs by upregulation of HDAC2 (histone deacetylase 2) expression and promotion of cell cycle proteins cyclin A1 and D1 expressions. Arginine supplementation did not affect LAP3 and HDAC2 expressions, but slowed down cell cycle process of malignant BMECs. In clinical samples of patients with breast cancer, LAP3 was confirmed to be upregulated, while ASS1 was downregulated compared with healthy control. CONCLUSIONS: These results demonstrated that LAP3 mediated IFN-γ-induced arginine depletion to malignant transformation of BMECs. Our findings provide a potential therapeutic target for breast cancer both in humans and dairy cows.

Regulated lytic cell death in breast cancer.

Breast cancer (BC) is a very common cancer among women and one of the primary causes of death in women worldwide. Because BC has different molecular subtypes, the challenges associated with targeted therapy have increased significantly, and the identification of new therapeutic targets has become increasingly urgent. Blocking apoptosis and inhibiting cell death are important characteristics of malignant tumours, including BC. Under adverse conditions, including exposure to antitumour therapy, inhibition of cell death programmes can promote cancerous transformation and the survival of cancer cells. Therefore, inducing cell death in cancer cells is fundamentally important and provides new opportunities for potential therapeutic interventions. Lytic forms of cell death, primarily pyroptosis, necroptosis and ferroptosis, are different from apoptosis owing to their characteristic lysis, that is, the production of cellular components, to guide beneficial immune responses, and the application of lytic cell death (LCD) in the field of tumour therapy has attracted considerable interest from researchers. The latest clinical research results confirm that lytic death signalling cascades involve the BC cell immune response and resistance to therapies used in clinical practice. In this review, we discuss the current knowledge regarding the various forms of LCD, placing a special emphasis on signalling pathways and their implications in BC, which may facilitate the development of novel and optimal strategies for the clinical treatment of BC.

IFN-γ-/- Mice Resist Actinobacillus pleuropneumoniae Infection by Promoting Early Lung IL-18 Release and PMN-I Accumulation.

Porcine pleuropneumonia is a common infectious disease of pigs caused by Actinobacillus pleuropneumoniae Interferon gamma (IFN-γ) expression increases in the lung of pigs after A. pleuropneumoniae infection, but the role of IFN-γ during the infection is still obscure. In this study, an IFN-γ-/- mouse infection model was established, and bacterial load, levels of inflammatory cytokines, and types of neutrophils in the lungs were studied at different times post-A. pleuropneumoniae infection. We found that wild-type (WT) mice were more susceptible to A. pleuropneumoniae than IFN-γ-/- mice. At 6 h postinfection (hpi), the expression of interleukin 18 (IL-18) and IL-1ß in the lungs of IFN-γ-/- mice was significantly increased compared to WT mice. The bacterial load and levels of inflammatory cytokines (IL-1ß and IL-6) of IFN-γ-/- mice were significantly reduced at 12 hpi compared to WT mice. After an initial loss, the numbers of lung polymorphonuclear (PMN)-I cells dramatically increased in the lungs of IFN-γ-/- but not WT mice, whereas PMN-II cells continually decreased. Finally, in vivo administration of IL-18 significantly reduced clinical scores and bacterial load in the lungs of A. pleuropneumoniae-infected mice. This study identifies IFN-γ as a target for regulating the inflammatory response in the lung and provides a basis for understanding the course of clinical bacterial pneumonia and for the formulation of treatment protocols.

RPSA distribution and expression in tissues and immune cells of pathogen-infected mice.

AIMS: 40S ribosomal protein SA (RPSA), a component of the small ribosomal subunit, is a high-affinity receptor of laminin that is widely expressed in cells and involves in many biological processes. However, it hasn't been reported which tissues and cells may be targeted by RPSA-mediated pathogen regulation. Therefore, in this study, a gram-positive bacterium Streptococcus suis Type 2 (SS2), gram-negative bacterium Actinobacillus pleuropneumoniae (A.pleuropneumoniae), and porcine circovirus Type 2 (PCV2) were used to infect ICR mice. METHODS AND RESULTS: The effects of infection with the three pathogens on expression levels of RPSA in mouse tissues and peripheral blood immune cells were analysed by immunohistochemistry and flow cytometry. The results suggested that the pathological changes in mice infected with SS2 were mainly manifested as congestion and inflammatory infiltration in the meninges, lungs, hearts and livers. The mice infected with A.pleuropneumoniae or PCV2 showed lung lesions and mild hepatocyte degeneration, respectively. In uninfected mice, RPSA protein was expressed to various degrees in all tissues except the spleen. After SS2 infection for 3 d, the expression of RPSA in the liver and brain increased, while decreased significantly in the heart and duodenum. These results were corroborated on examining the correlation between RPSA expression and the process of SS2 infection, except that there was no significant difference between the expression levels in the heart at 1 d and 3 d. After A.pleuropneumoniae and PCV2 infection for 3 d, the expression of RPSA decreased in the heart, and brain, respectively. Additionally, under physiological conditions, RPSA expression in CD4+ T cells, CD8+ T cells, neutrophils, and macrophages in the peripheral blood of mice was higher than that in B cells and NK cells. After SS2 infection for 3 d, RPSA expression increased significantly in CD4+ T cells and CD8+ T cells but decreased significantly in macrophages. The expression of RPSA after A.pleuropneumoniae and PCV2 infection were similar, and RPSA expression decreased only in macrophages. CONCLUSIONS: The results revealed that RPSA showed different expression levels in tissues and immune cells due to different pathogens causing disease courses, suggesting different target tissues and target cells in RPSA-mediated pathogenesis after infection, which supports the systematic study of the pathogenesis of RPSA in infectious diseases.

Streptococcus suis serotype 2 enolase interaction with host brain microvascular endothelial cells and RPSA-induced apoptosis lead to loss of BBB integrity.

Host proteins interacting with pathogens are receiving more attention as potential therapeutic targets in molecular medicine. Streptococcus suis serotype 2 (SS2) is an important cause of meningitis in both humans and pigs worldwide. SS2 Enolase (Eno) has previously been identified as a virulence factor with a role in altering blood brain barrier (BBB) integrity, but the host cell membrane receptor of Eno and The mechanism(s) involved are unclear. This study identified that SS2 Eno binds to 40S ribosomal protein SA (RPSA) on the surface of porcine brain microvascular endothelial cells leading to activation of intracellular p38/ERK-eIF4E signalling, which promotes intracellular expression of HSPD1 (heat-shock protein family D member 1), and initiation of host-cell apoptosis, and increased BBB permeability facilitating bacterial invasion. This study reveals novel functions for the host-interactional molecules RPSA and HSPD1 in BBB integrity, and provides insight for new therapeutic strategies in meningitis.

The Phage Holin HolGH15 Exhibits Potential As an Antibacterial Agent to Control Listeria monocytogenes.

Listeria monocytogenes is an important foodborne pathogen that is a serious threat to public health security, and new strategies to control this bacterium in food are needed. HolGH15, derived from Staphylococcus aureus phage GH15, has shown antibacterial activity against several bacterial species. In this work, the antilisterial behavior and effectiveness of HolGH15 are further studied. To elucidate its antimicrobial modes against L. monocytogenes, cell integrity and membrane permeabilization assays were performed. When treated with HolGH15, the release of 260-nm-absorbing materials of L. monocytogenes was rapidly increased. HolGH15 triggered a significant increase in fluorescence intensity by flow cytometry. In membrane permeabilization assays, the cytoplasmic ß-galactosidase of L. monocytogenes treated with HolGH15 was released via an increase in the permeability of the membrane. HolGH15 caused changes in the structural properties of L. monocytogenes cells resulting in shrinkage, which evoked the release and removal of cellular contents and finally lead to cell death. Electron microscopy observations indicated that HolGH15 exhibited excellent bactericidal potency by permeabilizing the cell membrane, damaging membrane integrity, and inducing cellular content shrinkage or loss. Moreover, HolGH15 (at the final concentration of 240 µg/mL) reduced L. monocytogenes (at the initial concentration of 106 colony-forming unit/mL) to an undetectable level at 4°C. Collectively, HolGH15 has potential as a novel antimicrobial agent against L. monocytogenes in the manufacture and store of food by spraying or soaking, especially at refrigerated temperature.

Necroptosis and its role in infectious diseases.

Necroptosis is a noncaspase-dependent and precisely regulated mechanism of cell death. Necroptosis is mainly initiated by members of the tumor necrosis factor receptor (TNFR) and Toll-like receptor (TLR) families, interferon, intracellular RNA and DNA sensors and other mediators. Subsequently, the protein kinase RIPK1 (receptor-interacting protein kinase 1) and RIPK3 interact with the receptor protein, which transduces death signals and further recruits and phosphorylates MLKL (mixed lineage kinase domain-like protein). MLKL serves as the initiator of cell death and eventually induces necroptosis. It was found that necroptosis is not only involved in the physiological regulation but also in the occurrence, development and prognosis of some necrotic diseases, especially infectious diseases. Intervention in the necroptosis signaling pathway is helpful for removing pathogens, inhibiting the development of lesions, and promoting the remodeling of tissue. In-depth study of the molecular regulation mechanism of necroptosis and its relationship with the pathogenesis of infectious diseases will help to provide new ideas and directions for research of the pathological mechanisms and clinical prevention of infectious diseases.

Therapeutic Efficacy of Phage PIZ SAE-01E2 against Abortion Caused by Salmonella enterica Serovar Abortusequi in Mice.

Salmonella enterica subsp. enterica serovar Abortusequi is a frequently reported pathogen causing abortion in mares. In this study, the preventive and therapeutic effects of phage PIZ SAE-01E2 against S Abortusequi in a mouse model of abortion were investigated. Phage PIZ SAE-01E2 was stable at different temperatures (4 to 70°C) and pH values (pH 4 to 10) and could lyse the majority of the Salmonella serogroup O:4 and O:9 strains tested (25/28). There was no lysogeny-related, toxin, or antibiotic resistance-related gene in the genome of PIZ SAE-01E2. All of these characteristics indicate that PIZ SAE-01E2 has the potential for use in phage therapy. In in vivo experiments, 2 × 103 CFU/mouse of S Abortusequi ATCC 9842 was sufficient to lead to murine abortion (gestational day 14.5) within 48 h. A single intraperitoneal inoculation of PIZ SAE-01E2 (108 PFU/mouse, multiplicity of infection = 105) 1 h before or after S Abortusequi challenge provided effective protection to all pregnant mice (10/10). After 24 h of treatment with phage PIZ SAE-01E2, the bacterial loads in both the placenta and the uterus of the infected mice were significantly decreased (<102 CFU/g) compared to those in the placenta and the uterus of the mice in the control group (>106 CFU/g). In addition, the levels of inflammatory cytokines in the placenta and blood of the mice in the phage administration groups were significantly reduced (P < 0.05) compared to those in the placenta and blood of the mice in the control group. Altogether, these findings indicate that PIZ SAE-01E2 shows the potential to block abortions induced by S Abortusequi in vivoIMPORTANCES Abortusequi is an important pathogen that can induce abortions in mares. Although S Abortusequi has been well controlled in Europe and the United States due to strict breeding and health policies, it is still widespread in African and Asian countries and has proven difficult to control. In China, abortions caused by S Abortusequi have also been reported in donkeys. So far, there is no commercial vaccine. Thus, exploiting alternative efficient and safe strategies to control S Abortusequi infection is essential. In this study, a new lytic phage, PIZ SAE-01E2, infecting S Abortusequi was isolated, and the characteristics of PIZ SAE-01E2 indicated that it has the potential for use in phage therapy. A single intraperitoneal inoculation of PIZ SAE-01E2 before or after S Abortusequi challenge provided effective protection to all pregnant mice. Thus, PIZ SAE-01E2 showed the potential to block abortions induced by S Abortusequi in vivo.

Therapeutic applications of lytic phages in human medicine.

The emergence and spread of antibiotic-resistant bacteria constitute a critical issue for modern medicine. Patients with antibiotic-resistant bacterial infections consume more healthcare resources and have worse clinical outcomes than patients with antibiotic-sensitive bacterial infections. Phages are natural predators of bacteria and may therefore be a source of useful antibacterial drugs. Phage therapy possess availability for oral administration, penetration through the bacteria cell wall, and eradication bacterial biofilms. All of these advantages give phage therapy the possibility to turn into applications for infectious diseases. In this mini-review, we focus on the brief history of lytic phage therapy, the life cycles of lytic phages and the therapeutic effects of lytic phages.

miR-31 shuttled by halofuginone-induced exosomes suppresses MFC-7 cell proliferation by modulating the HDAC2/cell cycle signaling axis.

Traditional Chinese medicine (TCM) are both historically important therapeutic agents and important source of new drugs. Halofuginone (HF), a small molecule alkaloid derived from febrifugine, has been shown to exert strong antiproliferative effects that differ markedly among various cell lines. However, whether HF inhibits MCF-7 cell growth in vitro and underlying mechanisms of this process are not yet clear. Here, we offer the strong evidence of the connection between HF treatment, exosome production and proliferation of MCF-7 cells. Our results showed that HF inhibits MCF-7 cell growth in both time- and dose-dependent manner. Further microRNA (miRNA) profiles analysis in HF treated and nontreated MCF-7 cell and exosomes observed that six miRNAs are particularly abundant and sorted in exosomes. miRNAs knockdown experiment in exosomes and the MCF-7 growth inhibition assay showed that exosomal microRNA-31 (miR-31) modulates MCF-7 cells growth by specially targeting the histone deacetylase 2 (HDAC2), which increases the levels of cyclin-dependent kinases 2 (CDK2) and cyclin D1 and suppresses the expression of p21. In conclusion, these data indicate that inhibition of exosome production reduces exosomal miR-31, which targets the HDAC2 and further regulates the level of cell cycle regulatory proteins, contributing to the anticancer functions of HF. Our data suggest a new role for HF and the exosome production in tumorigenesis and may provide novel insights into prevention and treatment of breast cancer.

Establishment and comparison of Actinobacillus pleuropneumoniae experimental infection model in mice and piglets.

Actinobacillus pleuropneumoniae (APP) causes porcine pleuropneumonia, a disease responsible for substantial losses in the worldwide pig industry. In this study, outbred Kunming (KM) and Institute of Cancer Research (ICR) mice were evaluated as alternative mice models for APP research. After intranasal infection of serotype 5 reference strain L20, there was less lung damage and a lower clinical sign score in ICR compared to KM mice. However, ICR mice showed more obvious changes in body weight loss, the amount of immune cells (such as neutrophils and lymphocytes) and cytokines (such as IL-6, IL-1ß and TNF-α) in blood and bronchoalveolar lavage fluid (BALF). The immunological changes observed in ICR mice closely mimicked those found in piglets infected with L20. While both ICR and KM mice are susceptible to APP and induce pathological lesions, we suggest that ICR and KM mice are more suitable for immunological and pathogenesis studies, respectively. The research lays the theoretical basis for determine that mice could replace pigs as the APP infection model and it is of significance for the study of APP infection in the laboratory.

Biological properties and genomics analysis of vB_KpnS_GH-K3, a Klebsiella phage with a putative depolymerase-like protein.

Bacteriophages have been recently revisited as an alternative biocontrol tool due to the limitations of antibiotic treatment. In this study, we reported on the biological characteristics and genomic information of vB_KpnS_GH-K3 (abbreviated as GH-K3), a Klebsiella phage of the Siphoviridae family, which was previously isolated from a hospital sewage system. One-step growth curve analysis indicated that the burst size of GH-K3 was 291 PFU/cell. GH-K3 maintained a stable titer in a broad range of pH values (6-10) and temperature (up to 50 °C). Based on bioinformatics analysis, GH-K3 comprises of 49,427 bp containing a total of 77 open reading frames (ORFs), which share high degree of nucleotide similarity and close evolutionary relationships with at least 12 other Klebsiella phages. Of note, GH-K3 gp32 was identified as a unique ORF. The major segment of gp32 sequence at the C-terminus (residues 351-907) was found highly variable as determined by its mismatch with the nucleotide and protein sequences available at NCBI database. Furthermore, HHpred analysis indicated that GH-K3 gp32 contains three domains (PDB ID: 5W6S_A, 3GQ8_A and 1BHE_A) similar to depolymerase (depoKP36) of Klebsiella phage KP36 suggestive of a potential depolymerase activity during host receptor-binding in the processes of phage infection. Altogether, the current data revealed a novel putative depolymerase-like protein which is most likely to play an important role in phage-host interaction.

Arginine inhibits the malignant transformation induced by interferon-gamma through the NF-κB-GCN2/eIF2α signaling pathway in mammary epithelial cells in vitro and in vivo.

Breast cancer is the most common female malignant tumors in the world. It seriously affects women's physical and mental health and the leading cause of cancer death among women. Our previous study demonstrated that diet-derived IFN-γ promoted the malignant transformation of primary bovine mammary epithelial cells by accelerating arginine depletion. The current study aimed to explore whether arginine addition could inhibit the degree of malignant transformation and its molecular mechanism. The results indicate that arginine addition could alleviate the malignant transformation of mammary epithelial cells induced by IFN-γ, including reducing cell proliferation, cell migration and colony formation, through the NF-κB-GCN2/eIF2α pathway. The in vivo experiments also consistently confirmed that arginine supplementation could significantly inhibit tumor growth in tumor-bearing mice. Furthermore, the investigation of the clinical data also revealed that the plasma or tissue from human breast cancer patients owned lower arginine level and higher IFN-γ level than that from patients with benign breast disease, showing IFN-γ may be a potential control target. Our findings demonstrate that arginine supplement could antagonize the malignant transformation of mammary epithelial cells induced by IFN-γ (nutritionally induced) both in vitro and in vivo, and IFN-γ was higher in breast cancer women. This might provide a novel strategy for the prevention and treatment of breast cancer regarding to nutrition.

A Smooth-Type, Phage-Resistant Klebsiella pneumoniae Mutant Strain Reveals that OmpC Is Indispensable for Infection by Phage GH-K3.

Bacteriophage can be used as an alternative or complementary therapy to antibiotics for treating multidrug-resistant bacterial infections. However, the rapid emergence of resistant host variants during phage treatment has limited its therapeutic applications. In this study, a potential phage-resistant mechanism of Klebsiella pneumoniae was revealed. After phage GH-K3 treatment, a smooth-type colony, named K7RB, was obtained from the K. pneumoniae K7 culture. Treatment with IO4- and/or proteinase K indicated that polysaccharides of K7 played an important role in phage recruitment, and protein receptors on K7 were essential for effective infection by GH-K3. Differences in protein expression between K7 and K7RB were quantitatively analyzed by liquid chromatography-tandem mass spectrometry. Among differentially expressed proteins, OmpC, OmpN, KPN_02430, and OmpF were downregulated significantly in K7RBtrans-Complementation of OmpC in K7RB conferred rapid adsorption and sensitivity to GH-K3. In contrast, a single-base deletion mutation of ompC in K7, which resulted in OmpC silencing, led to lower adsorption efficiency and resistance to GH-K3. These assays proved that OmpC is the key receptor-binding protein for GH-K3. In addition, the native K. pneumoniae strains KPP14, KPP27, and KPP36 showed low or no sensitivity to GH-K3. However, these strains became more sensitive to GH-K3 after their native receptors were replaced by OmpC of K7, suggesting that OmpCK7 was the most suitable receptor for GH-K3. This study revealed that K7RB became resistant to GH-K3 due to gene mutation of ompC and that OmpC of K7 is essential for effective infection by GH-K3.IMPORTANCE With increased incidence of multidrug-resistant (MDR) bacterial strains, phages have regained attention as promising potential antibacterial agents. However, the rapid emergence of resistant variants during phage treatment has limited the therapeutic applications of phage. According to our trans-complementation, ompC mutation, and phage adsorption efficiency assays, we identified OmpC as the key receptor-binding protein (RBP) for phage GH-K3, which is essential for effective infection. This study revealed that the phage secondary receptor of K. pneumoniae, OmpC, is the essential RBP not only for phage infecting Gram-negative bacteria, such as Escherichia coli and Salmonella, but also for K. pneumoniae.

Antibacterial Effects of Phage Lysin LysGH15 on Planktonic Cells and Biofilms of Diverse Staphylococci.

Treatment of infections caused by staphylococci has become more difficult because of the emergence of multidrug-resistant strains as well as biofilm formation. In this study, we observed the ability of the phage lysin LysGH15 to eliminate staphylococcal planktonic cells and biofilms formed by Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis All these strains were sensitive to LysGH15, showing reductions in bacterial counts of approximately 4 log units within 30 min after treatment with 20 µg/ml of LysGH15, and the MICs ranged from 8 µg/ml to 32 µg/ml. LysGH15 efficiently prevented biofilm formation by the four staphylococcal species at a dose of 50 µg/ml. At a higher dose (100 µg/ml), LysGH15 also showed notable disrupting activity against 24-h and 72-h biofilms formed by S. aureus and coagulase-negative species. In the in vivo experiments, a single intraperitoneal injection of LysGH15 (20 µg/mouse) administered 1 h after the injection of S. epidermidis at double the minimum lethal dose was sufficient to protect the mice. The S. epidermidis cell counts were 4 log units lower in the blood and 3 log units lower in the organs of mice 24 h after treatment with LysGH15 than in the untreated control mice. LysGH15 reduced cytokine levels in the blood and improved pathological changes in the organs. The broad antistaphylococcal activity exerted by LysGH15 on planktonic cells and biofilms makes LysGH15 a valuable treatment option for biofilm-related or non-biofilm-related staphylococcal infections.IMPORTANCE Most staphylococcal species are major causes of health care- and community-associated infections. In particular, Staphylococcus aureus is a common and dangerous pathogen, and Staphylococcus epidermidis is a ubiquitous skin commensal and opportunistic pathogen. Treatment of infections caused by staphylococci has become more difficult because of the emergence of multidrug-resistant strains as well as biofilm formation. In this study, we found that all tested S. aureus, S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis strains were sensitive to the phage lysin LysGH15 (MICs ranging from 8 to 32 µg/ml). More importantly, LysGH15 not only prevented biofilm formation by these staphylococci but also disrupted 24-h and 72-h biofilms. Furthermore, the in vivo efficacy of LysGH15 was demonstrated in a mouse model of S. epidermidis bacteremia. Thus, LysGH15 exhibits therapeutic potential for treating biofilm-related or non-biofilm-related infections caused by diverse staphylococci.