The rise of multi-drug-resistant (MDR) bacteria has spurred renewed interest in the use of bacteriophages in therapy. However, mechanisms contributing to phage-mediated bacterial clearance in an ...animal host remain unclear. We investigated the effects of host immunity on the efficacy of phage therapy for acute pneumonia caused by MDR Pseudomonas aeruginosa in a mouse model. Comparing efficacies of phage-curative and prophylactic treatments in healthy immunocompetent, MyD88-deficient, lymphocyte-deficient, and neutrophil-depleted murine hosts revealed that neutrophil-phage synergy is essential for the resolution of pneumonia. Population modeling of in vivo results further showed that neutrophils are required to control both phage-sensitive and emergent phage-resistant variants to clear infection. This “immunophage synergy” contrasts with the paradigm that phage therapy success is largely due to bacterial permissiveness to phage killing. Lastly, therapeutic phages were not cleared by pulmonary immune effector cells and were immunologically well tolerated by lung tissues.
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•Efficacious phage therapy to pulmonary P. aeruginosa requires innate immune components•Neutrophils are required to control phage-sensitive and emergent phage-resistant bacteria•Models predict “immunophage synergy” arises due to nonlinear feedback
The mechanisms underlying phage-mediated bacterial clearance in an animal host remain unclear. By coupling animal experiments and in silico modeling, Roach et al. show that host innate immunity is essential for the efficacy of phages in treating respiratory bacterial infections, defining the concept of “immunophage synergy.”
In a previous study, six virulent bacteriophages PAK_P1, PAK_P2, PAK_P3, PAK_P4, PAK_P5 and CHA_P1 were evaluated for their in vivo efficacy in treating Pseudomonas aeruginosa infections using a ...mouse model of lung infection. Here, we show that their genomes are closely related to five other Pseudomonas phages and allow a subdivision into two clades, PAK_P1-like and KPP10-like viruses, based on differences in genome size, %GC and genomic contents, as well as number of tRNAs. These two clades are well delineated, with a mean of 86% and 92% of proteins considered homologous within individual clades, and 25% proteins considered homologous between the two clades. By ESI-MS/MS analysis we determined that their virions are composed of at least 25 different proteins and electron microscopy revealed a morphology identical to the hallmark Salmonella phage Felix O1. A search for additional bacteriophage homologs, using profiles of protein families defined from the analysis of the 11 genomes, identified 10 additional candidates infecting hosts from different species. By carrying out a phylogenetic analysis using these 21 genomes we were able to define a new subfamily of viruses, the Felixounavirinae within the Myoviridae family. The new Felixounavirinae subfamily includes three genera: Felixounalikevirus, PAK_P1likevirus and KPP10likevirus. Sequencing genomes of bacteriophages with therapeutic potential increases the quantity of genomic data on closely related bacteriophages, leading to establishment of new taxonomic clades and the development of strategies for analyzing viral genomes as presented in this article.
Abstract Viruses are ubiquitous and can infect any of the three existing cellular lineages (Archaea, Bacteria and Eukarya). Despite the persisting negative public perception of these entities, ...scientists learnt how to domesticate some of them. The study of molecular mechanisms essential to the completion of viral cycles has greatly contributed to deciphering fundamental processes in biology. Nowadays, viruses have entered the biotechnological era and numerous applications have already been developed. Viral-derived tools are used to manipulate genetic information, detect, diagnose, control and cure infectious diseases, or even design new structural assemblies. With the recent advances in the field of metagenomics, an overwhelming amount of information on novel viruses has become available. As current tools have been historically developed from a limited number of viruses, the potential of discoveries from new archaeal, bacterial and eukaryotic viruses may be limited only by our understanding of the multiple facets of viral cycles.
In this study, we describe the characterization, cloning, expression and purification of the lysin A gene of the mycobacteriophage TM4. The gene TM4_gp29 (gp29) is a 1644-bp gene that codes for a ...58.6-kDa protein and contains peptidoglycan recognition protein, Zn-binding and amidase catalytic domains. The gene was cloned into Escherichia coli using the 'His-Tag' pQE60 vector. After affinity chromatography-mediated purification, the protein was concentrated and visualized using sodium dodecyl sulphate polyacrylamide gel electrophoresis. Evidence of peptidoglycan-degrading activity was observed initially by a chloroform assay and later by conventional zymogram analysis.
Multidrug resistance (MDR) is a major obstacle to successful cancer chemotherapy. A typical form of MDR is due to the overexpression of membrane transport proteins., such as Glycoprotein-P (P-gp), ...resulting in an increased drug efflux preventing drug cytotoxicity. P-gp is mainly localized on the plasma membrane; however, it can also be endocytosed resulting in the trafficking of P-gp in endoplasmic reticulum, Golgi, endosomes, and lysosomes. The lysosomal P-gp has been found to be capable of transporting and sequestering P-gp substrates (e.g., Doxorubicin (Dox)) into lysosomes to protect cells against cytotoxic drugs. Many translational studies have shown that low-density lipoprotein receptor-related protein-1 (LRP-1) is involved in endocytosis and regulation of signalling pathways. LRP-1 mediates the endocytosis of a diverse set of extracellular ligands that play important roles in tumor progression. Here, we investigated the involvement of LRP-1 in P-gp expression and subcellular redistribution from the cell surface to the lysosomal membrane by endocytosis and its potential implication in P-gp-mediated multidrug resistance in MCF-7 cells. Our results showed that MCF-7 resistant cells (MCF-7R) overexpressed the P-gp, LRP-1 and LAMP-1 and were 11.66-fold resistant to Dox. Our study also revealed that in MCF-7R cells, lysosomes were predominantly high density compared to sensitized cells and P-gp was localized in the plasma membrane and lysosomes. LRP-1 blockade reduced lysosomes density and level of LAMP-1 and P-gp. It also affected the subcellular distribution of P-gp. Under these conditions, we restored Dox nuclear uptake and ERK 1/2 activation thus leading to MCF-7R cell sensitization to Dox. Our data suggest that LRP-1 is able to modulate the P-gp expression and subcellular redistribution by endocytosis and to potentiate the P-gp-acquired Dox resistance.
"Your mission should you choose to accept, is to locate the enemy, silently dock on the cell surface and inject the genetic blueprint. Once inside, you must then hijack the host's cellular machinery ...and assemble multiple clones. Working quickly then, slice open the cell wall perimeter and escape to repeat the process on the remaining enemy hosts" Although farfetched, this passage describes the efficient killing machinery of bacteriophages, and depicts the events which occur millions of times in nature every day as bacteria succumb to their tiny deadly nemeses.
Given their potential as specific and natural biocontrol agents, bacteriophages and their associated proteins have become the focus of renewed attention over the last decade. The aim of this study ...was to use a comparative modelling approach to generate a predicted 3D structure for LysB; a 332 amino acid lipolytic enzyme encoded by the mycobacteriophage Ardmore. The GXSXG pentapeptide, characteristic of lipolytic enzymes, was located at amino acid position 166-170. The three absolutely conserved residues among mycobacteriophage LysB proteins were also identified in Ardmore LysB as Ser-168, Gly-203 and Pro-205. CATH analysis of Ardmore LysB revealed a mainly Beta classification, Beta Barrel architecture and a topology similar to maltoporin. This is unlike the α/β hydrolase structure reported for the D29 LysB protein and, in fact appears in only 3 other sequenced LysB homologues to date. A search for conserved motifs within the amino acid sequence of LysB revealed the presence of both a cutinase motif and a PE-PPE motif. This study presents an in silico 3D predictive model of Ardmore lysin and confirms the high diversity of mycobacteriophages LysB proteins both at the sequence (2D) and structural (predicted 3D) levels.
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