Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be ...predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage-specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.
Virtually all studies of phage infections investigate bacteria growing exponentially in rich media. In nature, however, phages largely encounter non-growing cells. Bacteria entering stationary phase ...often activate well-studied stress defense mechanisms that drastically alter the cell, facilitating its long-term survival. An understanding of phage-host interactions in such conditions is of major importance from both an ecological and therapeutic standpoint. Here, we show that bacteriophage T4 can efficiently bind to, infect and kill E. coli in stationary phase, both in the presence and absence of a functional stationary-phase sigma factor, and explore the response of T4-infected stationary phase cells to the addition of fresh nutrients 5 or 24 h after that infection. An unexpected new mode of response has been identified. "Hibernation" mode is a persistent but reversible dormant state in which the infected cells make at least some phage enzymes, but halt phage development until appropriate nutrients become available before producing phage particles. Our evidence indicates that the block in hibernation mode occurs after the middle-mode stage of phage development; host DNA breakdown and the incorporation of the released nucleotides into phage DNA indicate that the enzymes of the nucleotide synthesizing complex, under middle-mode control, have been made and assembled into a functional state. Once fresh glucose and amino acids become available, the standard lytic infection process rapidly resumes and concentrations of up to 10(11) progeny phage (an average of about 40 phage per initially present cell) are produced. All evidence is consistent with the hibernation-mode control point lying between middle mode and late mode T4 gene expression. We have also observed a "scavenger" response, where the infecting phage takes advantage of whatever few nutrients are available to produce small quantities of progeny within 2 to 5 h after infection. The scavenger response seems able to produce no more than an average of one phage per originally available cell, and few if any further progeny are produced by cells in this mode even if fresh nutrients are made available later.
Bacteriophages recognize their host cells with the help of tail fiber and tailspike proteins that bind, cleave, or modify certain structures on the cell surface. The spectrum of ligands to which the ...tail fibers and tailspikes can bind is the primary determinant of the host range. Bacteriophages with multiple tailspike/tail fibers are thought to have a wider host range than their less endowed relatives but the function of these proteins remains poorly understood. Here, we describe the structure, function, and substrate specificity of three tailspike proteins of bacteriophage CBA120—TSP2, TSP3 and TSP4 (orf211 through orf213, respectively). We show that tailspikes TSP2, TSP3 and TSP4 are hydrolases that digest the O157, O77, and O78 Escherichia coli O-antigens, respectively. We demonstrate that recognition of the E. coli O157:H7 host by CBA120 involves binding to and digesting the O157 O-antigen by TSP2. We report the crystal structure of TSP2 in complex with a repeating unit of the O157 O-antigen. We demonstrate that according to the specificity of its tailspikes TSP2, TSP3, and TSP4, CBA120 can infect E. coli O157, O77, and O78, respectively. We also show that CBA120 infects Salmonella enterica serovar Minnesota, and this host range expansion is likely due to the function of TSP1. Finally, we describe the assembly pathway and the architecture of the TSP1–TSP2–TSP3–TSP4 branched complex in CBA120 and its related ViI-like phages.
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Phage therapy is the application of bacteria-specific viruses with the goal of reducing or eliminating pathogenic or nuisance bacteria. While phage therapy has become a broadly relevant technology, ...including veterinary, agricultural, and food microbiology applications, it is for the treatment or prevention of human infections that phage therapy first caught the world's imagination--see, especially, Arrowsmith by Sinclair Lewis (1925)--and which today is the primary motivator of the field. Nonetheless, though the first human phage therapy took place in the 1920s, by the 1940s the field, was in steep decline despite early promise. The causes were at least three-fold: insufficient understanding among researchers of basic phage biology; over exuberance, which led, along with ignorance, to carelessness; and the advent of antibiotics, an easier to handle as well as highly powerful category of antibacterials. The decline in phage therapy was neither uniform nor complete, especially in the former Soviet Republic of Georgia, where phage therapy traditions and practice continue to this day. In this review we strive toward three goals: 1. To provide an overview of the potential of phage therapy as a means of treating or preventing human diseases; 2. To explore the phage therapy state of the art as currently practiced by physicians in various pockets of phage therapy activity around the world, including in terms of potential commercialization; and 3. To avert a recapitulation of phage therapy's early decline by outlining good practices in phage therapy practice, experimentation, and, ultimately, commercialization.
Infections involving diabetic foot ulcers (DFU) are a major public health problem and have a substantial negative impact on patient outcomes. Osteomyelitis in an ulcerated foot substantially ...increases the difficulty of successful treatment. While literature suggests that osteomyelitis in selected patients can sometimes be treated conservatively, with no, or minimal removal of bone, we do not yet have clear treatment guidelines and the standard treatment failure fallback remains amputation. The authors report on the successful treatment, with a long term follow up, of a 63 YO diabetic female with distal phalangeal osteomyelitis using bacteriophage, a form of treatment offering the potential for improved outcomes in this era of escalating antibiotic resistance and the increasingly recognized harms associated with antibiotic therapy.
We here present detailed descriptions of successful treatment of a series of diabetic toe ulcers using the Eliava BioPreparations' commercial preparation of the very well-studied anti-staphylococcal ...bacteriophage Sb-1. This chapter outlines what we feel is an appropriate mechanism to speed movement toward full-scale clinical trials with bacteriophage use to treat wound infections and to help address the crisis in antibiotic resistance.
The mechanisms by which bacteriophage T4 converts the metabolism of its
host to one dedicated to progeny phage production was the subject of decades of intense research in many labs from the 1950s ...through the 1980s. Presently, a wide range of phages are starting to be used therapeutically and in many other applications, and also the range of phage sequence data available is skyrocketing. It is thus important to re-explore the extensive available data about the intricacies of the T4 infection process as summarized here, expand it to looking much more broadly at other genera of phages, and explore phage infections using newly-available modern techniques and a range of appropriate environmental conditions.
Purpose
The potential of aerosol phage therapy for treating lung infections has been demonstrated in animal models and clinical studies. This work compared the performance of two dry powder formation ...techniques, spray freeze drying (SFD) and spray drying (SD), in producing inhalable phage powders.
Method
A
Pseudomonas
podoviridae phage, PEV2, was incorporated into multi-component formulation systems consisting of trehalose, mannitol and L-leucine (F1 = 60:20:20 and F2 = 40:40:20). The phage titer loss after the SFD and SD processes and
in vitro
aerosol performance of the produced powders were assessed.
Results
A significant titer loss (~2 log) was noted for droplet generation using an ultrasonic nozzle employed in the SFD method, but the conventional two-fluid nozzle used in the SD method was less destructive for the phage (~0.75 log loss). The phage were more vulnerable during the evaporative drying process (~0.75 log further loss) compared with the freeze drying step, which caused negligible phage loss.
In vitro
aerosol performance showed that the SFD powders (~80% phage recovery) provided better phage protection than the SD powders (~20% phage recovery) during the aerosolization process. Despite this, higher total lung doses were obtained for the SD formulations (SD-F1 = 13.1 ± 1.7 × 10
4
pfu and SD-F2 = 11.0 ± 1.4 × 10
4
pfu) than from their counterpart SFD formulations (SFD-F1 = 8.3 ± 1.8 × 10
4
pfu and SFD-F2 = 2.1 ± 0.3 × 10
4
pfu).
Conclusion
Overall, the SD method caused less phage reduction during the powder formation process and the resulted powders achieved better aerosol performance for PEV2.
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