A novel phage putative depolymerase, Depo16, has specific activity against K1 capsular-type Klebsiella pneumoniae.

Klebsiella pneumoniae, especially hypervirulent K. pneumoniae (hvKP), is a common opportunistic pathogen that often causes hospital- and community-acquired infections. Capsular polysaccharide (CPS) is an important virulence factor of K. pneumoniae. Some phages encode depolymerases that can recognize and degrade bacterial polysaccharides. In this study, the lytic bacteriophage vB_KpnP_ZK1 (abbreviated as ZK1) was isolated using serotype K1 hvKP as the host. Although amino acid sequence BLAST analysis indicated that the tail fiber protein Depo16 of phage ZK1 showed no significant similarity to any reported phage depolymerases, it displayed enzymatic activities that are characteristic of phage depolymerases. After expression and purification, Depo16 could efficiently remove the capsular polysaccharide layer that surrounds the surface of serotype K1 K. pneumoniae. Although no bactericidal activity was detected, Depo16 makes serotype K1 K. pneumoniae sensitive to peritoneal macrophages (PMs). In addition, in a mouse bacteremia model of serotype K1 K. pneumoniae, 25 µg of Depo16 was effective in significantly prolonging survival. Depo16 treatment can reduce the bacterial load in blood and major tissues and alleviate tissue damage in mice. This indicates that the putative depolymerase Depo16 is a potential antibacterial agent against serotype K1 K. pneumoniae infections.IMPORTANCEKlebsiella pneumoniae often causes hospital-acquired infections and community-acquired infections. Capsular polysaccharide (CPS) is one of the crucial virulence factors of K. pneumoniae. K1 and K2 capsular-type K. pneumoniae strains are the most prevalent serotypes of hypervirulent K. pneumoniae (hvKP). In this study, a novel K. pneumoniae phage named vB_KpnP_ZK1 was isolated, and its putative depolymerase Depo16 showed low homology with other reported phage depolymerases. Depo16 can specifically degrade the K. pneumoniae K1 capsule making this serotype sensitive to peritoneal macrophages. More importantly, Depo16 showed a significant therapeutic effect in a mouse bacteremia model caused by serotype K1 K. pneumoniae. Thus, Depo16 is a potential antibacterial agent to combat serotype K1 K. pneumoniae infections.

Structural biology and functional features of phage-derived depolymerase Depo32 on Klebsiella pneumoniae with K2 serotype capsular polysaccharides.

Hypervirulent Klebsiella pneumoniae with capsular polysaccharides (CPSs) causes severe nosocomial- and community-acquired infections. Phage-derived depolymerases can degrade CPSs from K. pneumoniae to attenuate bacterial virulence, but their antimicrobial mechanisms and clinical potential are not well understood. In the present study, Klebsiella phage GH-K3-derived depolymerase Depo32 (encoded by gene gp32) was identified to exhibit high efficiency in specifically degrading the CPSs of K2 serotype K. pneumoniae. The cryo-electron microscopy structure of trimeric Depo32 at a resolution up to 2.32 Å revealed potential catalytic centers in the cleft of each of the two adjacent subunits. K. pneumoniae subjected to Depo32 became more sensitive to phagocytosis by RAW264.7 cells and activated the cells by the mitogen-activated protein kinase signaling pathway. In addition, intranasal inoculation with Depo32 (a single dose of 200 µg, 20 µg daily for 3 days, or in combination with gentamicin) rescued all C57BL/6J mice infected with a lethal dose of K. pneumoniae K7 without interference from its neutralizing antibody. In summary, this work elaborates on the mechanism by which Depo32 targets the degradation of K2 serotype CPSs and its potential as an antivirulence agent. IMPORTANCE Depolymerases specific to more than 20 serotypes of Klebsiella spp. have been identified, but most studies only evaluated the single-dose treatment of depolymerases with relatively simple clinical evaluation indices and did not reveal the anti-infection mechanism of these depolymerases in depth. On the basis of determining the biological characteristics, the structure of Depo32 was analyzed by cryo-electron microscopy, and the potential active center was further identified. In addition, the effects of Depo32 on macrophage phagocytosis, signaling pathway activation, and serum killing were revealed, and the efficacy of the depolymerase (single treatment, multiple treatments, or in combination with gentamicin) against acute pneumonia caused by Klebsiella pneumoniae was evaluated. Moreover, the roles of the active sites of Depo32 were also elucidated in the in vitro and in vivo studies. Therefore, through structural biology, cell biology, and in vivo experiments, this study demonstrated the mechanism by which Depo32 targets K2 serotype K. pneumoniae infection.

Antihypertensive activity of different components of Veratrum alkaloids through metabonomic data analysis.

BACKGROUND: Hypertension is a serious global public health issue. Blood pressure (BP) is still not effectively controlled in about 20 - 30% of hypertensive patients. Therefore, it is imperative to develop new treatments for hypertension. Veratrum alkaloids were once used for the clinical treatment of hypertension, the mechanism of which is still unclear. It was gradually phased out due to adverse reactions. PURPOSE: This study aimed to investigate the short-term and long-term hypotensive profiles of different components of Veratrum alkaloids in spontaneously hypertensive rats (SHRs) to unveil their mechanisms of action. RESULTS: Total Veratrum alkaloid (V), component A (A), and veratramine (M) quickly decreased BP within 30 min of treatment, reduced renal and cardiovascular damage, and improved relevant biochemical indicators (nitric oxide [NO], endothelin-1 [ET-1], angiotensin II [Ang II)], noradrenaline [NE], etc) in SHRs to delay stroke occurrence. Thereinto, A exhibited excellent protective effects in cardiovascular disease. The metabolomic profiles of SHRs treated with V, A, and M were significantly different from those of SHRs treated with vehicle. Thirteen metabolites were identified as potential pharmacodynamic biomarkers. Through Kyoto Encyclopedia of Genes and Genomes analysis, V, A, and M-induced hypotension was mainly related to alterations in nicotinate and nicotinamide metabolism, GABAergic synapses, linoleic acid metabolism, ketone body synthesis and degradation, arginine and proline metabolism, and urea cycle, of which nicotinate and nicotinamide metabolism was the key metabolic pathway to relieve hypertension. CONCLUSION: This work shows that A is an effective and promising antihypertensive agent for hypertension treatment to reduce BP and hypertensive target organ damage, which is mainly mediated through modulating nicotinate and nicotinamide metabolism, RAS, and NO-ET homeostasis.

Protective effect of Eucommia ulmoides Oliver male flowers on ethanol-induced DNA damage in mouse cerebellum and cerebral cortex.

Ethanol is a principal ingredient of alcoholic beverages with potential neurotoxicity and genotoxicity, and the ethanol-associated oxidative DNA damage in the central nervous system is well documented. Natural product may offer new options to protect the brain against ethanol-induced neurotoxicity. The male flower of Eucommia ulmoides (EUF) Oliver has been extensively utilized as the tea, the healthy hot drink on the market. In this study, 19 constituents in the effective fraction of EUF were identified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). In the single-cell gel electrophoresis assay, EUF was observed to ameliorate DNA damage in mouse cerebellum and cerebral cortex caused by acute ethanol administration, which was further confirmed by the morphological observation. The protective effects of EUF were associated with increasing total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-PX) activities, and a decrease in nitric oxide (NO), malondialdehyde (MDA), 8-hydroxy-2'-deoxyguanosine (8-OHdG), and kelch-like ECH-associated protein-1 (Keap1) levels. Molecular docking results demonstrated that compounds 4, 7, 9, and 16 from EUF have a strong affinity to the Keap1 Kelch domain to hinder the interaction of nuclear factor-erythroid 2-related factor 2 (Nrf2) with Keap1. These findings suggest that EUF is a potent inhibitor of ethanol-induced brain injury possibly via the inhibition of oxidative stress.

The Bacteriophage vB_CbrM_HP1 Protects Crucian Carp Against Citrobacter braakii Infection.

Citrobacter braakii is an opportunistic pathogen that induces aquatic infections in fish and turtles. In this study, a bacteriophage that infects C. braakii, named vB_CbrM_HP1, was isolated from sewage. This phage belongs to Myoviridae family, Ounavirinae subfamily, Mooglevirus genus. We also used the phage to treat crucian carp infection caused by C. braakii for the first time. vB_CbrM_HP1 was relatively stable at temperatures ranging from 4 to 60°C and pH values ranging from 3 to 11 but float slightly. When the multiplicities of infection (MOI) was 0.0001, the titer reached a maximum of 4.20 × 1010 PFU/ml. As revealed from the results of whole genomic sequence analysis, the total length of vB_CbrM_HP1 was 89335 bp, encoding 135 ORFs, 9 of which were <75% similar to the known sequences in NCBI. The phage vB_CbrM_HP1 showed a highly efficient bactericidal effect against C. braakii both in vitro and in vivo. In vitro, vB_CbrM_HP1 was capable of effectively killing bacteria (the colony count decreased by 4.7 log units at 5 h). In vivo, administration of vB_CbrM_HP1 (1 × 109 PFU) effectively protected crucian carp against fatal infection caused by C. braakii. Phage treatment reduced the levels of inflammatory factors. All these results demonstrated the potential of vB_CbrM_HP1 as an alternative treatment strategy for infections caused by C. braakii.

Phage resistance mutation triggered by OmpC deficiency in Klebsiella pneumoniae induced limited fitness costs.

Outer membrane proteins (OMPs) play an important role in bacterial fitness costs. Derived from the interaction between Klebsiella pneumoniae K7 and phage GH-K3, K7RB is an outer membrane porin-deficient phage-resistant mutant strain triggered by ompC712 deletion, exhibits expression inhibition of OmpC, OmpN, KPN_02430 and OmpF, but its fitness costs and regulatory mechanism remains unknown. In this study, compared with K7, K7RB showed almost unaffected growth rate, slightly decreased virulence, and increased resistance to some antibiotics. Transcriptome analysis showed that the pathways of glycerolipid metabolism and nitrogen metabolism in K7RB were significantly inhibited, while the transcription of permeases belonging to ABC transporters tended to be active, nutrient uptakes such as citrate and phenylalanine were also enhanced. However, transcriptional up-regulation in K7RB was inhibited by overexpression of OmpC, OmpN, KPN_02430 and OmpF in general. Overexpression of OmpN, KPN_02430 and OmpF, respectively, restoring the sensitivity of strains to antibiotics to varying degrees, while OmpC overexpression aggravated the bacterial drug-resistance especially to ß-lactam antibiotics. Besides, unlike OmpC and OmpF, overexpression of OmpN and KPN_02430 reduced bacterial virulence. In brief, by revealing the limited fitness costs of phage-resistant mutant K. pneumoniae with porin-deficiency, our study providing a reference for the design and development of drugs to inhibit the ways of bacterial metabolic rewiring and to increase fitness costs.

A novel lysin Ply1228 provides efficient protection against Streptococcus suis type 2 infection in a murine bacteremia model.

Streptococcus suis is an important zoonotic pathogen that is difficult to control with antibiotics due to the widespread development of multidrug-resistant strains. Phage lysin is considered a potential therapeutic agent to combat S. suis. In this study, the novel lysin Ply1228 derived from the prophage of S. suis type 12 was identified. Bioinformatics analysis showed that Ply1228 contains a CHAP catalytic domain, which is a binding domain composed of a CW-7 binding motif and an amidase-2 catalytic domain. The CHAP catalytic domain is essential for the bactericidal function of lysin Ply1228 and does not depend on the presence of Ca2+. C34 and H99 of the CHAP domain were identified as the key active sites. The CW-7 binding motif plays a key binding role in Ply1228. Ply1228 can specifically lyse S. suis, including types 2, 3, 7, 9, 10, 12, 14, and 27. Within 10 min, Ply1228 killed 4 log of the S. suis population, which had a starting concentration of approximately 107 CFU/mL. In addition, Ply1228 showed favourable thermal and pH stability. The therapeutic effect of Ply1228 was further investigated in a mouse model of S. suis bacteremia. The administration of the lysin Ply1228 (200 µg/mouse) 1 h after the intraperitoneal injection of 2 × MLD of SS2 strain SC225 was sufficient to protect the mice (P < 0.0001) and significantly reduced the bacterial loads in the blood and organs (livers, spleens, lungs and kidneys). The levels of inflammation and histopathological damage in infected mice were effectively relieved after the Ply1228 treatment. These results indicate that Ply1228 might represent a new enzybiotic candidate for S. suis infection.

Combination Therapy of Phage vB_KpnM_P-KP2 and Gentamicin Combats Acute Pneumonia Caused by K47 Serotype Klebsiella pneumoniae.

Klebsiella pneumoniae (K. pneumoniae) is an important nosocomial and community acquired opportunistic pathogen which causes various infections. The emergence of multi-drug resistant (MDR) K. pneumoniae and carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) has brought more severe challenge to the treatment of K. pneumoniae infection. In this study, a novel bacteriophage that specifically infects K. pneumoniae was isolated and named as vB_KpnM_P-KP2 (abbreviated as P-KP2). The biological characteristics of P-KP2 and the bioinformatics of its genome were analyzed, and then the therapeutic effect of P-KP2 was tested by animal experiments. P-KP2 presents high lysis efficiency in vitro. The genome of P-KP2 shows homology with nine phages which belong to "KP15 virus" family and its genome comprises 172,138 bp and 264 ORFs. Besides, P-KP2 was comparable to gentamicin in the treatment of lethal pneumonia caused by K. pneumoniae W-KP2 (K47 serotype). Furthermore, the combined treatment of P-KP2 and gentamicin completely rescued the infected mice. Therefore, this study not only introduces a new member to the phage therapeutic library, but also serves as a reference for other phage-antibiotic combinations to combat MDR pathogens.

Bacteriophage Protects Against Aerococcus viridans Infection in a Murine Mastitis Model.

Bovine mastitis, an inflammatory disease that occurs frequently in early lactation or the dry period, is primarily caused by bacterial infections. There is growing evidence that Aerococcus viridans (A. viridans) is becoming an important cause of bovine mastitis. The treatment of bovine mastitis is primarily based on antibiotics, which not only leads to a large economic burden but also the development of antibiotic resistance. On the other hand, bacteriophages present a promising alternative treatment strategy. The object of this study was to evaluate the potential of a previously isolated A. viridans phage vB_AviM_AVP (AVP) as an anti-mastitis agent in an experimental A. viridans-induced murine mastitis model. A. viridans N14 was isolated from the milk of clinical bovine mastitis and used to establish a mastitis model in mice. We demonstrated that administration of phage AVP significantly reduced colony formation by A. viridans and alleviated damage to breast tissue. In addition, reduced inflammation was indicated by decreased levels of inflammatory cytokines (TNF-α, IL-1ß, and IL-6) and myeloperoxidase (MPO) activity in the phage-treated group compared to those in the phosphate buffered saline (PBS)-treated group. To the best of our knowledge, this report is the first to show the potential use of phages as a treatment for A. viridans-induced mastitis.

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.

The Yersinia Phage X1 Administered Orally Efficiently Protects a Murine Chronic Enteritis Model Against Yersinia enterocolitica Infection.

Yersinia enterocolitica is generally considered an important food-borne pathogen worldwide, especially in the European Union. A lytic Yersinia phage X1 (Viruses; dsDNA viruses, no RNA stage; Caudovirales; and Myoviridae) was isolated. Phage X1 showed a broad host range and could effectively lyse 27/51 Y. enterocolitica strains covering various serotypes that cause yersiniosis in humans and animals (such as serotype O3 and serotype O8). The genome of this phage was sequenced and analyzed. No toxin, antibiotic-resistance or lysogeny related modules were found in the genome of phage X1. Studies of phage stability confirmed that X1 had a high tolerance toward a broad range of temperatures (4-60°C) and pH values (4-11) for 1 h. The ability to resist harsh acidic conditions and enzymatic degradation in vitro demonstrated that phage X1 is suitable for oral administration, and in particular, that this phage can pass the stomach barrier and efficiently reach the intestine in vivo without losing infectious ability. The potential of this phage against Y. enterocolitica infection in vitro was studied. In animal experiments, a single oral administration of phage X1 at 6 h post infection was sufficient to eliminate Y. enterocolitica in 33.3% of mice (15/45). In addition, the number of Y. enterocolitica strains in the mice was also dramatically reduced to approximately 103 CFU/g after 18 h compared with 107 CFU/g in the mice without phage treatment. Treatment with phage X1 showed significant improvement by intestinal histopathologic observations. Moreover, proinflammatory cytokine levels (IL-6, TNF-α, and IL-1ß) were significantly reduced (P < 0.05). These results indicate that phage X1 is a promising candidate to control infection by Y. enterocolitica in vivo.

Interplay between autophagy and apoptosis in selenium deficient cardiomyocytes in chicken.

Dietary selenium (Se) deficiency can cause heart dysfunction, however the exact mechanism remains unclear. To understand this mechanism, 180day-old chicks, divided into two groups, C (control group) and L (low Se group), were fed with either a Se-sufficient (0.23mg/kg) or Se-deficient (0.033mg/kg) diets for 25days, respectively. Heart tissues and blood samples were collected. In L group, the activities of serum creatine kinase (CK) and creatine kinase-myoglobin (CK-MB) increased and typical ultrastructural apoptotic features were observed. Se deficiency up-regulated the mRNA levels of Cysteinyl aspartate specific proteinase 3 (Caspase-3), Cysteinyl aspartate specific proteinase 8 (Caspase-8), Cysteinyl aspartate specific proteinase 9 (Caspase-9), B cell lymphoma/leukemia 2 (Bcl-2), Bcl-2 Associated X Protein (Bax), (P<0.05), whereas, the mRNA levels of Microtubuleassociated protein light chains 3-1 (LC3-1), Autophagy associated gene 5 (ATG-5), Mammalian target of rapamycin (mTOR), Dynein and Becline-1 were down-regulated (P<0.05). Noticeably, Microtubuleassociated protein light chains 3-2 (LC3-2) mRNA level increased (P<0.05) by 20%. Western blot results showed that Se deficiency decreased the expression of Becline-1 and LC3-1 protein, however, the expression of Bax, Caspase-3 and Cysteinyl aspartate specific proteinase 12 (Caspase-12) increased at protein levels. The present study revealed that Se deficiency induced apoptosis while inhibited autophagy in chicken cardiomyocytes through Bax/Bcl-2 inhibition and caspases-mediated cleavage of Becline-1. Moreover, correlation analysis illustrates that apoptosis and autophagy might function contradictorily. Altogether we conclude that Se deficient chicken cardiomyocytes experienced apoptosis rather than autophagy which is considered to be more pro-survival.