Complex interactions between bacteriophages and their bacterial hosts play significant roles in shaping the structure of environmental microbial communities, not only by genetic transduction but also by modification of bacterial gene expression patterns. Survival of phages solely depends on their ability to infect their bacterial hosts, most importantly during phage entry. Successful dynamic adaptation of bacteriophages when facing selective pressures, such as host adaptation and resistance, dictates their abundance and diversification. Co-evolution of the phage tail fibers and bacterial receptors determine bacterial host ranges, mechanisms of phage entry, and other infection parameters. This review summarizes the current knowledge about the physical interactions between tailed bacteriophages and bacterial pathogens (e.g., Salmonella enterica and Pseudomonas aeruginosa) and the influences of the phage on host gene expression. Understanding these interactions can offer insights into phage–host dynamics and suggest novel strategies for the design of bacterial pathogen biological controls.
Clostridium difficile is recognized as a problematic pathogen, causing severe enteric diseases including antibiotic-associated diarrhea and pseudomembranous colitis. The emergence of antibiotic resistant C. difficile has driven a search for alternative anti-infection modalities. A promising strategy for controlling bacterial infection includes the use of bacteriophages and their gene products. Currently, knowledge of phages active against C. difficile is still relatively limited by the fact that the isolation of phages for this organism is a technically demanding method since bacterial host themselves are difficult to culture. To isolate and characterize phages specific to C. difficile, a genotoxic agent, mitomycin C, was used to induce temperate phages from 12 clinical isolates of C. difficile. Five temperate phages consisting of ΦHR24, ΦHN10, ΦHN16-1, ΦHN16-2, and ΦHN50 were successfully induced and isolated. Spotting assays were performed against a panel of 92 C. difficile isolates to screen for susceptible bacterial hosts. The results revealed that all the C. difficile phages obtained in this work displayed a relatively narrow host range of 0–6.5% of the tested isolates. Electron microscopic characterization revealed that all isolated phages contained an icosahedral head connected to a long contractile tail, suggesting that they belonged to the Myoviridae family. Restriction enzyme analysis indicated that these phages possess unique double-stranded DNA genome. Further electron microscopic characterization revealed that the ΦHN10 absorbed to the bacterial surface via attachment to cell wall, potentially interacting with S-layer protein. Bacteriophages isolated from this study could lead to development of novel therapeutic agents and detection strategies for C. difficile.
For cryo-EM structural studies we seek to image membrane proteins as single particles embedded in proteoliposomes. One technical difficulty has been the low density of liposomes that can be trapped in the ~100 nm ice layer that spans holes in the perforated carbon support film of EM grids. Inspired by the use of two-dimensional (2D) streptavidin crystals as an affinity surface for biotinylated DNA (Crucifix et al., 2004), we propose to use the crystals to tether liposomes doped with biotinylated lipids. The 2D crystal image also serves as a calibration of the image formation process, providing an absolute conversion from electrostatic potentials in the specimen to the EM image intensity, and serving as a quality control of acquired cryo-EM images. We were able to grow streptavidin crystals covering more than 90% of the holes in an EM grid, and which remained stable even under negative stain. The liposome density in the resulting cryo-EM sample was uniform and high due to the high-affinity binding of biotin to streptavidin. Using computational methods, the 2D crystal background can be removed from images without noticeable effect on image properties.
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