Expression of a phage-encoded Gp21 protein protects Pseudomonas aeruginosa against phage infection

There is a continuously expanding gap between predicted phage gene sequences and their corresponding functions, which largely hampered the development of phage therapy. Previous studies reported several phage proteins that could interfere with the intracellular processes of the host to obtain efficient infection. But few phage proteins that protect host against phage infection has been identified and characterized in detail. Here, we isolate a phage vB_Pae_QDWS capable of infecting Pseudomonas aeruginosa PAO1, and report its encoded Gp21 protein protects PAO1 against phage infection. Expressing of Gp21 regulate bacterial quorum sensing with an inhibitory effect in low cell density and activation effect in high cell density. By testing the TFPs-mediated twitching motility and transmission electron microscopy analysis, Gp21 was found decreased the pilus synthesis. Further constructing the TFPs synthesis gene pilB mutant and performing adsorption and phage resistance assay, we demonstrated Gp21 protein could block phage infection via decreasing the TFPs-mediated phage adsorption. Gp21 is a novel protein that inhibit phage efficacy against bacteria. The study deepens our understanding of phage-host interactions. Importance The majority of the annotated phage genes are currently deposited as “hypothetical protein” with unknown function. Researches revealed that some phage proteins serve to inhibit or redirect the host intracellular processes for phage infection. Differently, we report a phage encoded protein Gp21 that protect the host against phage infection. The pathways that Gp21 involved in anti-phage defense in Pseudomonas aeruginosa PAO1 are interfering with quorum sensing and decreasing the type IV pilus-mediated phage adsorption. Gp21 is a novel protein with a low sequence homology with other reported twitching inhibitory proteins. As a lytic phage derived protein, Gp21 expression protects P. aeruginosa PAO1 from reinfection by phage vB_Pae_QDWS, which may explain the well-known pseudolysogeny caused by virulent phages. Our discoveries provide valuable new insight into the phage-host evolutionary dynamics.

Pseudo-188D: Phage Protein Prediction Based on a Model of Pseudo-188D

Phages have seriously affected the biochemical systems of the world, and not only are phages related to our health, but medical treatments for many cancers and skin infections are related to phages; therefore, this paper sought to identify phage proteins. In this paper, a Pseudo-188D model was established. The digital features of the phage were extracted by PseudoKNC, an appropriate vector was selected by the AdaBoost tool, and features were extracted by 188D. Then, the extracted digital features were combined together, and finally, the viral proteins of the phage were predicted by a stochastic gradient descent algorithm. Our model effect reached 93.4853%. To verify the stability of our model, we randomly selected 80% of the downloaded data to train the model and used the remaining 20% of the data to verify the robustness of our model.

Prediction and Analysis in silico of Genomic Islands in Aeromonas hydrophila

Aeromonas are Gram-negative rods widely distributed in the environment. They can cause severe infections in fish related to financial losses in the fish industry, and are considered opportunistic pathogens of humans causing infections ranging from diarrhea to septicemia. The objective of this study was to determine in silico the contribution of genomic islands to A. hydrophila. The complete genomes of 17 A. hydrophila isolates, which were separated into two phylogenetic groups, were analyzed using a genomic island (GI) predictor. The number of predicted GIs and their characteristics varied among strains. Strains from group 1, which contains mainly fish pathogens, generally have a higher number of predicted GIs, and with larger size, than strains from group 2 constituted by strains recovered from distinct sources. Only a few predicted GIs were shared among them and contained mostly genes from the core genome. Features related to virulence, metabolism, and resistance were found in the predicted GIs, but strains varied in relation to their gene content. In strains from group 1, O Ag biosynthesis clusters OX1 and OX6 were identified, while strains from group 2 each had unique clusters. Metabolic pathways for myo-inositol, L-fucose, sialic acid, and a cluster encoding QueDEC, tgtA5, and proteins related to DNA metabolism were identified in strains of group 1, which share a high number of predicted GIs. No distinctive features of group 2 strains were identified in their predicted GIs, which are more diverse and possibly better represent GIs in this species. However, some strains have several resistance attributes encoded by their predicted GIs. Several predicted GIs encode hypothetical proteins and phage proteins whose functions have not been identified but may contribute to Aeromonas fitness. In summary, features with functions identified on predicted GIs may confer advantages to host colonization and competitiveness in the environment.

Bacteriophage as an antibacterial agent: a patent perspective

The present review encompasses a patent landscape on bacteriophage as an antimicrobial agent and one of the alternatives to combat antibiotic resistance in bacteria. This study gives a perspective on use of bacteriophages in various industries such as healthcare, food safety and animal and plant protection. Patenting activity was noted for all the antibiotic-resistant bacterial pathogens listed in the ‘critical’ category by the WHO. Broadly, claims of the analyzed patents were directed toward bacteriophage, composition/formulation containing phage, phage proteins and various methods of using or producing phage. The challenges to approval of phage therapy in clinical use may be overcome with the help of focused research and modification of the regulatory guidelines for phage therapy.

Isolation and characterization of a lytic bacteriophage against Pseudomonas aeruginosa

In recent years, the use of bacteriophages (or 'phages') against multidrug-resistant (MDR) bacteria including Pseudomonas aeruginosa has drawn considerable attention, globally. In this work, we report the isolation and detailed characterization of a highly lytic Pseudomonasphage DRL-P1 isolated from wastewater. Under TEM, DRL-P1 appeared as a member of the phage family Myoviridae. DRL-P1 featured rapid adsorption (~ 5 min), short-latency (~ 30 min), and large burst size (~ 100 PFU per infected cell). DRL-P1 can withstand a wide temperature range (4 °C to 40 °C) and pH (5.0 to 10.0) conditions. The 66,243 bp DRL-P1 genome (MN564818) encodes at least 93 ORFs, of which 36 were functionally annotated based on homology with similar phage proteins available in the databases. Comparative analyses of related genomes suggest an independent evolutionary history and discrete taxonomic position of DRL-P1 within genus Pbunavirus. No toxin or antibiotic resistance genes was identified. DRL-P1 is tolerant to lyophilization and encapsulation techniques and retained lytic activity even after 18 months of storage. We also demonstrated decontaminating potentials of DRL-P1 in vitro, on an artificially contaminated cover-slip model. To the best of our knowledge, this is the first Pbunavirus to be reported from India. Our study suggests DRL-P1 as a potential candidate for various applications.

Influence of Caudovirales Phages on Humoral Immunity in Mice

Bacteriophages are promising antibacterial agents. Although they have been recognized as bacterial viruses and are considered to be non-interacting with eukaryotic cells, there is growing evidence that phages may have a significant impact on the immune system via interactions with macrophages, neutrophils, and T-cell polarization. In this study, the influence of phages of podovirus, siphovirus, and myovirus morphotypes on humoral immunity of CD-1 mice was investigated. In addition, tissue distribution of the phages was tested in these mice. No common patterns were found either in the distribution of phages in mice or in changes in the levels of cytokines in the sera of mice once injected with phages. Importantly, pre-existing IgM-class antibodies directed against capsid proteins of phages with myovirus and siphovirus morphotypes were identified in mice before immunization. After triple immunization of CD1-mice with phages without any adjuvant, levels of anti-phage serum polyclonal IgG antibodies increased. Immunogenic phage proteins recognized by IgM and/or IgG antibodies were identified using Western blot analysis and mass spectrometry. In addition, mice serum collected after immunization demonstrated neutralizing properties, leading to a substantial decrease in infectivity of investigated phages with myovirus and siphovirus morphotypes. Moreover, serum samples collected before administration of these phages exhibited some ability to reduce the phage infectivity. Furthermore, Proteus phage PM16 with podovirus morphotype did not elicit IgM or IgG antibodies in immunized mice, and no neutralizing activities against PM16 were revealed in mouse serum samples before and after immunization.

Molecular mechanism of quorum sensing inhibition in Streptococcus by the phage protein paratox

Streptococcus pyogenes, or Group A Streptococcus, is a Gram-positive bacterium that can be both a human commensal and pathogen. Central to this dichotomy are temperate bacteriophages that incorporate into the bacterial genome as a prophage. These genetic elements encode both the phage proteins as well as toxins harmful to the human host. One such conserved phage protein paratox (Prx) is always found encoded adjacent to the toxin genes and this linkage is preserved during transduction. Within Streptococcus pyogenes, Prx functions to inhibit the quorum-sensing ComRS receptor-signal pair that is the master regulator of natural competence, or the ability to uptake endogenous DNA. Specifically, Prx directly binds and inhibits the receptor ComR by unknown mechanism. To understand how Prx inhibits ComR at the molecular level we pursued an X-ray crystal structure of Prx bound to ComR. The structural data supported by solution X-ray scattering data demonstrate that Prx induces a conformational change in ComR to directly access the DNA binding domain. Furthermore, electromobility shift assays and competition binding assays reveal that Prx effectively uncouples the inter-domain conformational change that is required for activation of ComR by the signaling molecule XIP. Although to our knowledge the molecular mechanism of quorum-sensing inhibition by Prx is unique, it is analogous to the mechanism employed by the phage protein Aqs1 in Pseudomonas aeruginosa. Together, this demonstrates an example of convergent evolution between Gram-positive and Gram-negative phages to inhibit quorum-sensing, and highlights the versatility of small phage proteins.

Mutations in Ehrlichia chaffeensis Genes ECH_0660 and ECH_0665 Cause Transcriptional Changes in Response to Zinc or Iron Limitation

Ehrlichia chaffeensis causes human monocytic ehrlichiosis by replicating within phagosomes of monocytes/macrophages. A function disruption mutation within the pathogen’s ECH_0660 gene encoding a phage head-to-tail connector protein resulted in the rapid clearance of the pathogen in vivo, while aiding to induce sufficient immunity in a host to protect against wild-type infection challenge. In this study, we describe the characterization of a cluster of seven genes spanning from ECH_0659 to ECH_0665, which contained four genes encoding bacterial phage proteins, including the ECH_0660 gene. Assessment of the promoter region upstream to the first gene of the seven genes (ECH_0659) in Escherichia coli demonstrated transcriptional enhancement under zinc and iron starvation. Further, transcription of the seven genes was significantly higher for E. chaffeensis having a mutation in the ECH_0660 gene compared to the wild-type pathogen under zinc and iron starving conditions. In contrast, transcription from the genes was mostly similar to wild-type or moderately downregulated for the ECH_0665 gene mutant with the function disruption. Recently, we reported that this mutation caused a minimal impact on the pathogen’s in vivo growth, as it persisted similar to wild-type. The current study is the first in describing how zinc and iron contribute to E. chaffeensis biology. Specifically, we demonstrated that the functional disruption in the gene encoding the predicted head-to-tail connector protein in E. chaffeensis results in the enhanced transcription of seven genes including those encoding phage proteins during zinc and iron limitation. IMPORTANCE Ehrlichia chaffeensis, a tick-transmitted bacterium, causes human monocytic ehrlichiosis by replicating within phagosomes of monocytes/macrophages. A function disruption mutation within the pathogen’s gene encoding a phage head-to-tail connector protein resulted in the rapid clearance of the pathogen in vivo, while aiding to induce sufficient immunity in a host to protect against wild-type infection challenge. In the current study, we investigated if the functional disruption in the predicted head-to-tail connector protein gene caused transcriptional changes resulting from metal ion limitations. This is the first study describing how zinc and iron may contribute to E. chaffeensis replication.

Proteomic Characterization of Bacteriophage Peptides from the Mastitis Producer Staphylococcus aureus by LC-ESI-MS/MS and the Bacteriophage Phylogenomic Analysis

The present work describes LC-ESI-MS/MS MS (liquid chromatography-electrospray ionization-tandem mass spectrometry) analyses of tryptic digestion peptides from phages that infect mastitis-causing Staphylococcus aureus isolated from dairy products. A total of 1933 nonredundant peptides belonging to 1282 proteins were identified and analyzed. Among them, 79 staphylococcal peptides from phages were confirmed. These peptides belong to proteins such as phage repressors, structural phage proteins, uncharacterized phage proteins and complement inhibitors. Moreover, eighteen of the phage origin peptides found were specific to S. aureus strains. These diagnostic peptides could be useful for the identification and characterization of S. aureus strains that cause mastitis. Furthermore, a study of bacteriophage phylogeny and the relationship among the identified phage peptides and the bacteria they infect was also performed. The results show the specific peptides that are present in closely related phages and the existing links between bacteriophage phylogeny and the respective Staphylococcus spp. infected.

Comprehensive genomics depict accessory genes encoding pathogenicity and biofilm determinants in Enterococcus faecalis

Aim: Enterococcus faecalis is a leading nosocomial pathogen in biofilm-associated polymicrobial infections. The study aims to understand pathogenicity and biofilm determinants of the pathogen by genome analysis. Methodology: Genome sequencing of a strong biofilm forming clinical isolate Enterococcus faecalis SK460 devoid of Fsr quorum-signaling system, was performed and comparative genomics was carried out among a set of pathogenic biofilm formers and nonpathogenic weak biofilm formers. Results: Analysis revealed a pool of virulence and adhesion related factors associated with pathogenicity. Absence of CRISPR-Cas system facilitated acquisition of pheromone responsive plasmid, pathogenicity island and phages. Comprehensive analysis identified a subset of accessory genes encoding polysaccharide lyase, sugar phosphotransferase system, phage proteins and transcriptional regulators exclusively in pathogenic biofilm formers. Conclusion: The study identified a set of genes specific to pathogenic biofilm formers and these can act as targets which in turn help to develop future treatment endeavors against enterococcal infections.