RESUMO
Bacterial and food allergens are associated with immune-mediated food allergies via the gut-skin axis. However, there has been no data on the potential use of phages to rescue this pathological process. A human triple cell co-culture model incorporating colonocytes (T84 cells), macrophages (THP-1 cells), and hepatocytes (Huh7 cells) was established and infected with Pseudomonas aeruginosa PAO1 (P.a PAO1) in the absence or presence of its KPP22 phage in Dulbecco's Modified Eagle's Medium (DMEM), DMEM+ ovalbumin (OVA), or DMEM+ß-casein media. The physiological health of cells was verified by assessing cell viability and Transepithelial electrical resistance (TEER) across the T84 monolayer. The immune response of cells was investigated by determining the secretions of IL-1ß, IL-8, IL-22, and IL-25. The ability of P.a PAO1 to adhere to and invade T84 cells was evaluated. The addition of either OVA or ß-casein potentiated the P.a PAO1-elicited secretion of cytokines. The viability and TEER of the T84 monolayer were lower in the P.a PAO1+OVA group compared to the P.a PAO1 alone and PAO1+ß-casein groups. OVA and ß-casein significantly increased the adherence and invasion of P.a PAO1 to T84 cells. In the presence of the KPP22 phage, these disruptive effects were abolished. These results imply that: (1) food allergens and bacterial toxic effector molecules exacerbate each other's disruptive effects; (2) food allergen and bacterial signaling at the gut-skin mucosal surface axis depend on a network of bacteria-phage-eukaryotic host interactions; and (3) phages are complementary for the evaluation of pathobiological processes that occur at the interface between bacteria, host cellular milieu, and food antigens because phages intervene in P.a PAO1-, OVA-, and ß-casein-derived inflammation.
Assuntos
Alérgenos , Hipersensibilidade Alimentar , Humanos , Alérgenos/imunologia , Hipersensibilidade Alimentar/imunologia , Hipersensibilidade Alimentar/metabolismo , Pseudomonas aeruginosa/fisiologia , Bacteriófagos/fisiologia , Pele/imunologia , Pele/virologia , Pele/microbiologia , Citocinas/metabolismo , Técnicas de CoculturaRESUMO
Staphylococcus aureus (S. aureus), particularly Methicillin-resistant S. aureus (MRSA), poses a significant global public health threat, necessitating advanced methodologies to enhance our understanding of this organism at the omics levels. This study introduces a refined protocol for constructing and curing high-density transposon mutant (tn-mutant) libraries in S. aureus, addressing the challenges associated with low transductant yields, and the complex genetic manipulation mechanism in Gram-positive bacteria. Our methodology employs a Himar1 transposon based on a two-plasmid system, leveraging Himar1's high insertional efficiency in AT-rich organisms. Enhanced transduction efficiency was achieved through chloramphenicol pre-treatment and the use of modified enriched media. Complementing this, an optimized plasmid curing procedure ensured a representative and stable tn-mutant library. The protocol was successfully applied to multiple S. aureus strains, demonstrating an increase in mutant recovery and reduced post-curing impact. The method offers a robust approach for Transposon Insertion Sequencing (TIS) applications in S. aureus, enabling deeper insights into survival, resistance, and pathogenicity mechanisms. This protocol holds a significant potential for accelerating the construction of tn-mutant libraries in various S. aureus strains.
Assuntos
Elementos de DNA Transponíveis , Biblioteca Gênica , Mutagênese Insercional , Staphylococcus aureus , Elementos de DNA Transponíveis/genética , Staphylococcus aureus/genética , Mutagênese Insercional/métodos , Mutação , Plasmídeos/genética , Bacteriófagos/genética , Temperatura Alta , Staphylococcus aureus Resistente à Meticilina/genéticaRESUMO
Background: Klebsiella pneumoniae is a major cause of hospital-acquired infections (HAIs), primarily spread through environmental contamination in hospitals. The effectiveness of current chemical disinfectants is waning due to emerging resistance, which poses environmental hazards and fosters new resistance in pathogens. Developing environmentally friendly and effective disinfectants against multidrug-resistant organisms is increasingly important. Methods: This study developed a bacteriophage cocktail targeting two common carbapenem-resistant Klebsiella pneumoniae (CRKP) strains, ST11 KL47 and ST11 KL64. The cocktail was used as an adjunctive disinfectant in a hospital's respiratory intensive care unit (RICU) via ultrasonic nebulization. Digital PCR was used to quantify CRKP levels post-intervention. The microbial community composition was analyzed via 16S rRNA sequencing to assess the intervention's impact on overall diversity. Results: The phage cocktail significantly reduced CRKP levels within the first 24 hours post-treatment. While a slight increase in pathogen levels was observed after 24 hours, they remained significantly lower than those treated with conventional disinfectants. 16S rRNA sequencing showed a decrease in the target pathogens' relative abundance, while overall species diversity remained stable, confirming that phages selectively target CRKP without disrupting ecological balance. Discussion: The findings highlight the efficacy and safety of phage-based biocleaners as a sustainable alternative to conventional disinfectants. Phages selectively reduce multidrug-resistant pathogens while preserving microbial diversity, making them a promising tool for infection control.
Assuntos
Bacteriófagos , Descontaminação , Unidades de Terapia Intensiva , Klebsiella pneumoniae , RNA Ribossômico 16S , RNA Ribossômico 16S/genética , Klebsiella pneumoniae/virologia , Klebsiella pneumoniae/genética , Descontaminação/métodos , Bacteriófagos/genética , Humanos , Reação em Cadeia da Polimerase/métodos , Infecção Hospitalar/prevenção & controle , Infecção Hospitalar/microbiologia , Desinfetantes/farmacologia , Infecções por Klebsiella/prevenção & controle , Infecções por Klebsiella/microbiologia , Análise de Sequência de DNARESUMO
The increase of antibiotic-resistant bacteria has become a global health emergency and the need to explore alternative therapeutic options arises. Phage therapy uses bacteriophages to target specific bacterial strains. Phages are highly specific and can target resistant bacteria. Currently, research in this regard is focused on ensuring reliability and safety to bring this tool into clinical practice. The first step is to conduct comprehensive preclinical research. In this work, we present two novel bacteriophages vB_Kpn_F13 and vB_Kpn_F14 isolated against clinical carbapenem-resistant Klebsiella pneumoniae strains obtained from hospital sewage. Multiple studies in vitro were conducted, such as sequencing, electron microscopy, stability, host range infectivity, planktonic effect and biofilm inhibition in order to discover their ability to be used against carbapenem-resistant K. pneumoniae pathogens causing difficult-to-treat infections.
Assuntos
Bacteriófagos , Biofilmes , Enterobacteriáceas Resistentes a Carbapenêmicos , Carbapenêmicos , Infecções por Klebsiella , Klebsiella pneumoniae , Terapia por Fagos , Klebsiella pneumoniae/virologia , Klebsiella pneumoniae/efeitos dos fármacos , Bacteriófagos/isolamento & purificação , Bacteriófagos/fisiologia , Bacteriófagos/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Enterobacteriáceas Resistentes a Carbapenêmicos/virologia , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/terapia , Carbapenêmicos/farmacologia , Biofilmes/crescimento & desenvolvimento , Biofilmes/efeitos dos fármacos , Humanos , Especificidade de Hospedeiro , Esgotos/virologia , Esgotos/microbiologia , Antibacterianos/farmacologia , Genoma Viral , Testes de Sensibilidade MicrobianaRESUMO
Background: The infant gut microbiome's establishment is pivotal for health and immune development. Understanding it unveils insights into growth, development, and maternal microbial interactions. Research often emphasizes gut bacteria, neglecting the phageome. Methods: To investigate the influence of geographic or maternal factors (mode of delivery, mode of breastfeeding, gestational diabetes mellitus) on the gut microbiota and phages of newborns, we collected fecal samples from 34 pairs of mothers and their infants within 24 hours of delivery from three regions (9 pairs from Enshi, 7 pairs from Hohhot, and 18 pairs from Hulunbuir) using sterile containers. Gut microbiota analysis by Shotgun sequencing was subsequently performed. Results: Our results showed that geographic location affects maternal gut microbiology (P < 0.05), while the effect on infant gut microbiology was not significant (P = 0.184). Among the maternal factors, mode of delivery had a significant (P < 0.05) effect on the newborn. Specific bacteria (e.g., Bacteroides, Escherichia spp., Phocaeicola vulgatus, Escherichia coli, Staphylococcus hominis, Veillonella spp.), predicted active metabolites, and bacteriophage vOTUs varied with delivery mode. Phocaeicola vulgatus significantly correlated with some metabolites and bacteriophages in the early infant gut (P < 0.05). In the GD group, a strong negative correlation of phage diversity between mother and infants was observed (R = -0.58, P=0.04). Conclusion: In conclusion, neonatal early gut microbiome (including bacteria and bacteriophages) colonization is profoundly affected by the mode of delivery, and maternal gestational diabetes mellitus. The key bacteria may interact with bacteriophages to influence the levels of specific metabolites. Our study provides new evidence for the study of the infant microbiome, fills a gap in the analysis of the infant gut microbiota regarding the virome, and emphasizes the importance of maternal health for the infant initial gut virome.
Assuntos
Bactérias , Diabetes Gestacional , Fezes , Microbioma Gastrointestinal , Humanos , Diabetes Gestacional/microbiologia , Gravidez , Feminino , Recém-Nascido , Fezes/microbiologia , Fezes/virologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Adulto , Bacteriófagos/genética , Parto Obstétrico , Aleitamento MaternoRESUMO
Vibrio parahaemolyticus is a major seafood-borne zoonotic pathogen that causes gastroenteritis in humans and acute hepatopancreatic necrosis disease (AHPND) in shrimp. In this study, we isolated and characterized Vibrio phage vB_VpM-pA2SJ1, which infects clinical and AHPND-associated strains of V. parahaemolyticus. The phage genome is a linear dsDNA 51,054 bp in length with a G + C content of 43.7%, and it contains 89 open reading frames. Genome comparisons revealed basal similarity to other Vibrio phages, particularly Vibrio phage vB_VpP_1, with 84.2% identity and 46% coverage. Phylogenetic analysis based on the whole genome, the terminase large subunit, and the major capsid protein revealed that phage vB_VpM-pA2SJ1 did not cluster with other known phage families, thus indicating its uniqueness.
Assuntos
Bacteriófagos , Composição de Bases , Genoma Viral , Fases de Leitura Aberta , Filogenia , Vibrio parahaemolyticus , Vibrio parahaemolyticus/virologia , Vibrio parahaemolyticus/genética , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Bacteriófagos/classificação , Animais , Penaeidae/virologia , Penaeidae/microbiologia , Vibrioses/microbiologia , Vibrioses/virologia , Vibrioses/veterinária , Hepatopâncreas/virologia , Hepatopâncreas/microbiologia , Hepatopâncreas/patologia , DNA Viral/genéticaRESUMO
Traditional assays for counting bacteriophages and their lysogens are labor-intensive and perturbative to the host cells. Here, we present a high-throughput infection method in a microplate reader, where the growth dynamics of the infected culture is measured using the optical density (OD). We find that the OD at which the culture lyses scales linearly with the logarithm of the initial phage concentration, providing a way of measuring phage numbers over nine orders of magnitude and down to single-phage sensitivity. Interpreting the measured dynamics using a mathematical model allows us to infer the phage growth rate, which is a function of the phage-cell encounter rate, latent period, and burst size. Adding antibiotic selection provides the ability to measure the rate of host lysogenization. Using this method, we found that when E. coli growth slows down, the lytic growth rate of lambda phages decreases, and the propensity for lysogeny increases, demonstrating how host physiology influences the viral developmental program.
Assuntos
Escherichia coli , Lisogenia , Escherichia coli/virologia , Escherichia coli/crescimento & desenvolvimento , Bacteriófagos/fisiologia , Bacteriófago lambda/fisiologia , Bacteriófago lambda/genética , Modelos Biológicos , Dinâmica Populacional , Antibacterianos/farmacologia , Modelos TeóricosRESUMO
Two recent studies in Cell Host & Microbe (Cury et al. and van den Berg et al.) uncover cross-kingdom links between antiphage and antiviral immune defenses. Through reciprocal computational and wet lab approaches, they each discover and experimentally validate proteins used for host immunity.
Assuntos
Interações Hospedeiro-Patógeno , Humanos , Interações Hospedeiro-Patógeno/imunologia , Bacteriófagos/fisiologia , Bacteriófagos/imunologia , Biologia Computacional/métodos , Antivirais/imunologiaRESUMO
The isolation and identification of pathogenic bacteria from a variety of samples are critical for controlling bacterial infection-related health problems. The conventional methods, such as plate counting and polymerase chain reaction-based approaches, tend to be time-consuming and reliant on specific instruments, severely limiting the effective identification of these pathogens. In this study, we employed the specificity of the cell wall-binding (CBD) domain of the Staphylococcus aureus bacteriophage 80 alpha (80α) endolysin towards the host bacteria for isolation. Amidase 3-CBD conjugated magnetic beads successfully isolated as few as 1 × 102 CFU/mL of S. aureus cells from milk, blood, and saliva. The cell wall hydrolyzing activity of 80α endolysin promoted the genomic DNA extraction efficiency by 12.7 folds on average, compared to the commercial bacterial genomic DNA extraction kit. Then, recombinase polymerase amplification (RPA) was exploited to amplify the nuc gene of S. aureus from the extracted DNA at 37 °C for 30 min. The RPA product activated Cas12a endonuclease activity to cleave fluorescently labeled ssDNA probes. We then converted the generated signal into a fluorescent readout, detectable by either the naked eye or a portable, self-assembled instrument with ultrasensitivity. The entire procedure, from isolation to identification, can be completed within 2 h. The simplicity and sensitivity of the method developed in this study make it of great application value in S. aureus detection, especially in areas with limited resource supply.
Assuntos
Técnicas Biossensoriais , Endopeptidases , Staphylococcus aureus , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/virologia , Técnicas Biossensoriais/métodos , Endopeptidases/química , Endopeptidases/isolamento & purificação , Endopeptidases/genética , Bacteriófagos/química , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Humanos , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/química , Fagos de Staphylococcus/isolamento & purificação , Animais , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções Estafilocócicas/microbiologia , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Nuclease do Micrococo/química , Nuclease do Micrococo/metabolismo , Nuclease do Micrococo/genética , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
The toxicity and safety of a veterinary anti-salmonella disinfectant based on three highly virulent bacteriophage strains (titers 1010 PFU/ml) were studied. Acute, chronic, and inhalation toxicity, as well as local irritancy of the disinfectant were evaluated on outbred white mice CD1 (n=65), Soviet chinchilla rabbits (n=20), and rats (n=20). No toxic effects of the disinfectant was observed after its intraperitoneal or intragastric administration to mice and intragastric administration to rats; in rabbits, application on the skin and eyes produced no local irritation effect. Inhalation of 10% of the disinfectant did not cause any pathologies in mice. Thus, the tests confirmed the high level of safety of the disinfectant based on a mixture of bacteriophages for use as an additional specific disinfection agent against Salmonella in veterinary and livestock facilities.
Assuntos
Desinfetantes , Animais , Camundongos , Coelhos , Desinfetantes/farmacologia , Desinfetantes/toxicidade , Ratos , Bacteriófagos , Salmonella/efeitos dos fármacos , Feminino , Masculino , ChinchilaRESUMO
The bacteriophage F8 belongs to the Myoviridae group of phages and is a pathogen of Pseudomonas aeruginosa. Since Pseudomonas aeruginosa is a multidrug-resistant opportunistic bacterium and can cause serious challenges for health services, studying the potential use of phages against them is a promising approach. Pseudomonas aeruginosa can be found on medical devices because bacteria can attach to surfaces and develop biofilms, which are difficult to eradicate because of their high resistance to environmental conditions and antimicrobial therapeutics. Phage therapy is becoming promising as an alternative for the treatment of antibiotic-resistant infections, but there is still a lack of standardized protocols approved by health organizations for possible use in the clinic. In our research, we focused on the potential use of 1-octanol, which was previously used by our team to develop a method for phage purification from bacterial lysate. 1-octanol is a fatty alcohol that is mostly used in the cosmetics industry, and its advantage is that it is approved by the FDA as a food additive. In this paper, we studied the protective properties of the addition of 1-octanol for storing phage liquid preparations. We demonstrated the stabilization effect of 1-octanol addition on F8 bacteriophage preparation during storage under various conditions. Interestingly, more effective biofilm reduction was observed after treatment with the purified bacteriophage and with 1-octanol addition compared to crude lysate.
Assuntos
Biofilmes , Interações Hidrofóbicas e Hidrofílicas , Fagos de Pseudomonas , Pseudomonas aeruginosa , Pseudomonas aeruginosa/virologia , Biofilmes/crescimento & desenvolvimento , Fagos de Pseudomonas/fisiologia , 1-Octanol/química , Myoviridae/fisiologia , Myoviridae/química , Bacteriófagos/fisiologia , Bacteriófagos/químicaRESUMO
In two recent studies in Nature, Hör et al.1 and Chambers et al.2 report that ubiquitin-like conjugation in bacteria antagonizes phage replication.
Assuntos
Ubiquitinação , Ubiquitina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Bacteriófagos/metabolismo , Bacteriófagos/fisiologia , Replicação Viral , Bactérias/metabolismo , Bactérias/genética , Bactérias/virologiaRESUMO
Outer membrane vesicles (OMVs) are nano-sized vesicles actively released by Gram-negative bacteria, playing a crucial role in bacterial survival and interactions with phages. This review focuses on OMVs and succinctly delineates the stimuli instigating OMV formation, their functional repertoire, and their involvement in bacterial-phage interplays. Initially, the discussion centers on the drivers prompting OMV genesis, encompassing both extrinsic environmental pressures and intrinsic regulatory mechanisms within bacterial systems. Subsequently, a comprehensive examination of OMVs' multifaceted functions in bacterial physiology ensues, spanning signaling cascades, nutrient transport, antibiotic resilience, and evasion of immune surveillance. Particular emphasis is placed on elucidating the paramount significance of OMVs in mediating bacterial-phage dynamics. OMVs function as decoys, providing protection to bacterial hosts against phages, and concurrently promoting the spread of phage receptors, thereby rendering phage-resistant strains susceptible to phage invasion. This comprehensive review deepens our comprehension of membrane vesicles biogenesis in bacteria and their pivotal role in microbial community dynamics.
Assuntos
Bacteriófagos , Bacteriófagos/fisiologia , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/fisiologia , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Negativas/virologia , Bactérias/virologia , Bactérias/metabolismo , Vesículas Extracelulares/metabolismo , Fenômenos Fisiológicos BacterianosRESUMO
The increasing interest in bacteriophage technology has prompted its novel applications to treat different medical conditions, most interestingly cancer. Due to their high specificity, manipulability, nontoxicity, and nanosize nature, phages are promising carriers in targeted therapy and cancer immunotherapy. This approach is particularly timely, as current challenges in cancer research include damage to healthy cells, inefficiency in targeting, obstruction by biological barriers, and drug resistance. Some cancers are being kept at the forefront of phage research, such as colorectal cancer and HCC, while others like lymphoma, cervical cancer, and myeloma have not been retouched in a decade. Common mechanisms are immunogenic antigen display on phage coats and the use of phage as transporters to carry drugs, genes, and other molecules. To date, popular phage treatments being tested are gene therapy and phage-based vaccines using M13 and λ phage, with some vaccines having advanced to human clinical trials. The results from most of these studies have been promising, but limitations in phage-based therapies such as reticuloendothelial system clearance or diffusion inefficiency must be addressed. Before phage-based therapies for cancer can be successfully used in oncology practice, more in-depth research and support from local governments are required.
Assuntos
Neoplasias , Terapia por Fagos , Humanos , Neoplasias/terapia , Neoplasias/imunologia , Terapia por Fagos/métodos , Bacteriófagos/fisiologia , Imunoterapia/métodos , Animais , Terapia Genética/métodos , Vacinas Anticâncer/uso terapêutico , Vacinas Anticâncer/imunologiaRESUMO
Highly diverse phages infecting thermophilic bacteria of the Thermus genus have been isolated over the years from hot springs around the world. Many of these phages are unique, rely on highly unusual developmental strategies, and encode novel enzymes. The variety of Thermus phages is clearly undersampled, as evidenced, for example, by a paucity of phage-matching spacers in Thermus CRISPR arrays. Using water samples collected from hot springs in the Kunashir Island from the Kuril archipelago and from the Tsaishi and Nokalakevi districts in the Republic of Georgia, we isolated several distinct phages infecting laboratory strains of Thermus thermophilus. Genomic sequence analysis of 11 phages revealed both close relatives of previously described Thermus phages isolated from geographically distant sites, as well as phages with very limited similarity to earlier isolates. Comparative analysis allowed us to predict several accessory phage genes whose products may be involved in host defense/interviral warfare, including a putative Type V CRISPR-cas system.
Assuntos
Bacteriófagos , Genoma Viral , Fontes Termais , Filogenia , Thermus thermophilus , Thermus thermophilus/virologia , Thermus thermophilus/genética , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Bacteriófagos/classificação , Bacteriófagos/fisiologia , Fontes Termais/microbiologia , Fontes Termais/virologia , Sistemas CRISPR-Cas , República da Geórgia , Genômica/métodosRESUMO
Bacteriophages have been proposed as biological controllers to protect plants against different bacterial pathogens. In this scenario, one of the main challenges is the low viability of phages in plants and under adverse environmental conditions. This work explores the use of 12 compounds and 14 different formulations to increase the viability of a phage mixture that demonstrated biocontrol capacity against Pseudomonas syringae pv. actinidiae (Psa) in kiwi plants. The results showed that the viability of the phage mixture decreases at 44 °C, at a pH lower than 4, and under UV radiation. However, using excipients such as skim milk, casein, and glutamic acid can prevent the viability loss of the phages under these conditions. Likewise, it was demonstrated that the use of these compounds prolongs the presence of phages in kiwi plants from 48 h to at least 96 h. In addition, it was observed that phages remained stable for seven weeks when stored in powder with skim milk, casein, or sucrose after lyophilization and at 4 °C. Finally, the phages with glutamic acid, sucrose, or skim milk maintained their antimicrobial activity against Psa on kiwi leaves and persisted within kiwi plants when added through roots. This study contributes to overcoming the challenges associated with the use of phages as biological controllers in agriculture.
Assuntos
Doenças das Plantas , Pseudomonas syringae , Pseudomonas syringae/virologia , Pseudomonas syringae/efeitos dos fármacos , Doenças das Plantas/virologia , Doenças das Plantas/prevenção & controle , Doenças das Plantas/microbiologia , Agricultura/métodos , Actinidia/química , Bacteriófagos/fisiologia , Viabilidade Microbiana/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Agentes de Controle Biológico/farmacologia , Excipientes/química , Excipientes/farmacologia , Folhas de Planta/virologia , Folhas de Planta/químicaRESUMO
Xanthomonas oryzae pv. oryzae (Xoo) is a significant bacterial pathogen responsible for outbreaks of bacterial leaf blight in rice, posing a major threat to rice cultivation worldwide. Effective management of this pathogen is crucial for ensuring rice yield and food security. In this study, we identified and characterized a novel Xoo phage, ZP3, isolated from diseased rice leaves in Zhejiang, China, which may offer new insights into biocontrol strategies against Xoo and contribute to the development of innovative approaches to combat bacterial leaf blight. Transmission electron microscopy indicated that ZP3 had a short, non-contractile tail. Genome sequencing and bioinformatic analysis showed that ZP3 had a double-stranded DNA genome with a length of 44,713 bp, a G + C content of 52.2%, and 59 predicted genes, which was similar to other OP1-type Xoo phages belonging to the genus Xipdecavirus. ZP3's endolysin LysZP was further studied for its bacteriolytic action, and the N-terminal transmembrane domain of LysZP is suggested to be a signal-arrest-release sequence that mediates the translocation of LysZP to the periplasm. Our study contributes to the understanding of phage-Xoo interactions and suggests that phage ZP3 and its endolysin LysZP could be developed into biocontrol agents against this phytopathogen.
Assuntos
Bacteriófagos , Genoma Viral , Oryza , Doenças das Plantas , Xanthomonas , Xanthomonas/virologia , Xanthomonas/efeitos dos fármacos , Bacteriófagos/genética , Bacteriófagos/fisiologia , Bacteriófagos/isolamento & purificação , Oryza/microbiologia , Oryza/virologia , Doenças das Plantas/microbiologia , Doenças das Plantas/virologia , Endopeptidases/farmacologia , Endopeptidases/genética , Endopeptidases/química , Endopeptidases/metabolismo , Filogenia , Folhas de Planta/virologia , Folhas de Planta/microbiologia , China , Genômica/métodosRESUMO
The gut microbiome is an emerging factor in preventing hypertension, yet the influence of gut bacteriophages, viruses infecting bacteria, on this condition remains unclear. Bacteriophage-bacteria interactions, which impact the gut microbiome, are influenced differentially by temperate and virulent bacteriophages. However, the standard technique for studying viral populations, viral-like particles (VLPs)-metagenomes, often overlook prophages, the intracellular stage of temperate bacteriophages, creating a knowledge gap. To address this, we investigated alterations in extracellular and intracellular bacteriophages, alongside bacterial populations, in the angiotensin II-hypertension model. We sequenced VLPs and bulk DNA from cecal-colonic samples collected from male C57BL/6J mice implanted with minipumps containing saline or angiotensin II. We assembled 106 bacterial and 816 viral genomes and found that gut viral and bacterial populations remained stable between hypertensive and normotensive mice. A higher number of temperate viruses were observed across all treatments. Although temperate viruses outnumbered virulent viruses, sequencing of both VLPs and bulk revealed that virions from virulent viruses were more abundant in the murine gut. We then evaluated the impact of low- and high-fiber intake on gut microbiome composition in the angiotensin II model. Fiber intake significantly influenced the gut microbiome composition and hypertension development. Mice receiving high-fiber had lower blood pressure, a higher bacterial-encoded carbohydrate-associated enzyme, and a higher total relative abundance of temperate viruses than those receiving low-fiber. Our findings suggest that phages are not associated with hypertension development in the angiotensin II model. However, they support a complex diet-bacteria/phage interaction that may be involved in blood pressure regulation.
Assuntos
Bactérias , Bacteriófagos , Fibras na Dieta , Modelos Animais de Doenças , Microbioma Gastrointestinal , Hipertensão , Camundongos Endogâmicos C57BL , Animais , Fibras na Dieta/administração & dosagem , Camundongos , Masculino , Hipertensão/virologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bacteriófagos/fisiologia , Bacteriófagos/genética , Angiotensina II/metabolismo , Genoma ViralRESUMO
Phages, the natural predators of bacteria, were discovered more than 100 years ago. However, increasing antimicrobial resistance rates have revitalized phage research. Methods that are more time-consuming and efficient than wet-laboratory experiments are needed to help screen phages quickly for therapeutic use. Traditional computational methods usually ignore the fact that phage-bacteria interactions are achieved by key genes and proteins. Methods for intraspecific prediction are rare since almost all existing methods consider only interactions at the species and genus levels. Moreover, most strains in existing databases contain only partial genome information because whole-genome information for species is difficult to obtain. Here, we propose a new approach for interaction prediction by constructing new features from key genes and proteins via the application of K-means sampling to select high-quality negative samples for prediction. Finally, we develop DeepPBI-KG, a corresponding prediction tool based on feature selection and a deep neural network. The results show that the average area under the curve for prediction reached 0.93 for each strain, and the overall AUC and area under the precision-recall curve reached 0.89 and 0.92, respectively, on the independent test set; these values are greater than those of other existing prediction tools. The forward and reverse validation results indicate that key genes and key proteins regulate and influence the interaction, which supports the reliability of the model. In addition, intraspecific prediction experiments based on Klebsiella pneumoniae data demonstrate the potential applicability of DeepPBI-KG for intraspecific prediction. In summary, the feature engineering and interaction prediction approaches proposed in this study can effectively improve the robustness and stability of interaction prediction, can achieve high generalizability, and may provide new directions and insights for rapid phage screening for therapy.
Assuntos
Bacteriófagos , Aprendizado Profundo , Bacteriófagos/genética , Bactérias/genética , Bactérias/virologia , Biologia Computacional/métodosRESUMO
Objective: To express and purify the phage depolymerase from hypervirulent Klebsiella pneumoniae (hvKp) serotype K1 and validate its function. Methods: Phage that infected serotype K1-type hvKp was isolated from hospital sewage. The biology and morphology of the phage were determined by plaque assay and transmission electron microscopy. The whole genome of the phage was sequenced by the Illumina HiSeq 2500 platform. The presence of depolymerase was determined by observing the plaque halo. Bioinformatic analysis and prokaryotic protein expression system were further used to predict and identify phage depolymerase. The depolymerase gene fragment was obtained by PCR and cloned into the pET28a expression vector, and the expression and purification of the depolymerase were completed in strain BL21. The depolymerase activities on the capsular polysaccharide of serotype K1-type hvKp clinical isolates were detected by plaque assay and low-speed centrifugation assay. Results: A lytic phage (phiA2) that infected serotype K1-type hvKp clinical isolate was isolated from hospital sewage. It was typical of the Caudovirales order and Autographiviridae family, and its whole genome was 43 526 bp in length and contained 51 coding domain sequences. The phage phiA2-derived depolymerase phiA2-dep was predicted, expressed and purified. The plaque assay and low-speed centrifugation assay indicated that the depolymerase phiA2-dep had good lytic activity on the capsular polysaccharide of serotype K1-type hvKp clinical isolates. Conclusion: Depolymerase phiA2-dep can specifically degrade the capsular polysaccharide of serotype K1-type hvKp, which has potential application value in treating bacterial infection.