Despite Spirochetales being a ubiquitous and medically important order of bacteria infecting both humans and animals, there is extremely limited information regarding their bacteriophages. Of the ...genus Treponema, there is just a single reported characterised prophage.
We applied a bioinformatic approach on 24 previously published Treponema genomes to identify and characterise putative treponemal prophages. Thirteen of the genomes did not contain any detectable prophage regions. The remaining eleven contained 38 prophage sequences, with between one and eight putative prophages in each bacterial genome. The prophage regions ranged from 12.4 to 75.1 kb, with between 27 and 171 protein coding sequences. Phylogenetic analysis revealed that 24 of the prophages formed three distinct sequence clusters, identifying putative myoviral and siphoviral morphology. ViPTree analysis demonstrated that the identified sequences were novel when compared to known double stranded DNA bacteriophage genomes.
In this study, we have started to address the knowledge gap on treponeme bacteriophages by characterising 38 prophage sequences in 24 treponeme genomes. Using bioinformatic approaches, we have been able to identify and compare the prophage-like elements with respect to other bacteriophages, their gene content, and their potential to be a functional and inducible bacteriophage, which in turn can help focus our attention on specific prophages to investigate further.
Proteus mirabilis forms dense crystalline biofilms on catheter surfaces that occlude urine flow, leading to serious clinical complications in long-term catheterized patients, but there are presently ...no truly effective approaches to control catheter blockage by this organism. This study evaluated the potential for bacteriophage therapy to control P. mirabilis infection and prevent catheter blockage. Representative in vitro models of the catheterized urinary tract, simulating a complete closed drainage system as used in clinical practice, were employed to evaluate the performance of phage therapy in preventing blockage. Models mimicking either an established infection or early colonization of the catheterized urinary tract were treated with a single dose of a 3-phage cocktail, and the impact on time taken for catheters to block, as well as levels of crystalline biofilm formation, was measured. In models of established infection, phage treatment significantly increased time taken for catheters to block (∼ 3-fold) compared to untreated controls. However, in models simulating early-stage infection, phage treatment eradicated P. mirabilis and prevented blockage entirely. Analysis of catheters from models of established infection 10 h after phage application demonstrated that phage significantly reduced crystalline biofilm formation but did not significantly reduce the level of planktonic cells in the residual bladder urine. Taken together, these results show that bacteriophage constitute a promising strategy for the prevention of catheter blockage but that methods to deliver phage in sufficient numbers and within a key therapeutic window (early infection) will also be important to the successful application of phage to this problem.
The canonical view of phage - bacterial interactions in dense, liquid cultures is that the phage will eliminate most of the sensitive cells; genetic resistance will then ascend to restore high ...bacterial densities. Yet there are various mechanisms by which bacteria may remain sensitive to phages but still attain high densities in their presence - because bacteria enter a transient state of reduced adsorption. Importantly, these mechanisms may be cryptic and inapparent prior to the addition of phage yet result in a rapid rebound of bacterial density after phage are introduced. We describe mathematical models of these processes and suggest how different types of this 'phenotypic' resistance may be elucidated. We offer preliminary in vitro studies of a previously characterized E. coli model system and Campylobacter jejuni illustrating apparent phenotypic resistance. As phenotypic resistance may be specific to the receptors used by phages, awareness of its mechanisms may identify ways of improving the choice of phages for therapy. Phenotypic resistance can also explain several enigmas in the ecology of phage-bacterial dynamics. Phenotypic resistance does not preclude the evolution of genetic resistance and may often be an intermediate step to genetic resistance.
Antibiotic resistance is an increasing global problem resulting from the pressure of antibiotic usage, greater mobility of the population, and industrialization. Many antibiotic resistance genes are ...believed to have originated in microorganisms in the environment, and to have been transferred to other bacteria through mobile genetic elements. Among others, β-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to β-lactam antibiotics is conferred by β-lactamase genes and penicillin-binding proteins, which are chromosomal- or plasmid-encoded, although there is little information available on the contribution of other mobile genetic elements, such as phages. This study is focused on three genes that confer resistance to β-lactam antibiotics, namely two β-lactamase genes (blaTEM and blaCTX-M9) and one encoding a penicillin-binding protein (mecA) in bacteriophage DNA isolated from environmental water samples. The three genes were quantified in the DNA isolated from bacteriophages collected from 30 urban sewage and river water samples, using quantitative PCR amplification. All three genes were detected in the DNA of phages from all the samples tested, in some cases reaching 104 gene copies (GC) of blaTEM or 102 GC of blaCTX-M and mecA. These values are consistent with the amount of fecal pollution in the sample, except for mecA, which showed a higher number of copies in river water samples than in urban sewage. The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin. blaTEM and blaCTX were detected in the DNA of the resistant clones after transfection. This study indicates that phages are reservoirs of resistance genes in the environment.
The bacterial type VI secretion system (T6SS) is an organelle that is structurally and mechanistically analogous to an intracellular membrane-attached contractile phage tail. Recent studies ...determined that a rapid conformational change in the structure of a sheath protein complex propels T6SS spike and tube components along with antibacterial and antieukaryotic effectors out of predatory T6SS+ cells and into prey cells. The contracted organelle is then recycled in an ATP-dependent process. T6SS is regulated at transcriptional and posttranslational levels, the latter involving detection of membrane perturbation in some species. In addition to directly targeting eukaryotic cells, the T6SS can also target other bacteria coinfecting a mammalian host, highlighting the importance of the T6SS not only for bacterial survival in environmental ecosystems, but also in the context of infection and disease. This review highlights these and other advances in our understanding of the structure, mechanical function, assembly, and regulation of the T6SS.
Ho et al. examine the structure, mechanical function, assembly, and regulation of the bacterial type VI secretion system.
Aims
Catheter‐associated urinary tract infections (CAUTI) caused by Proteus mirabilis are very difficult to treat due to the ability of biofilm formation and drug resistance of these bacteria. The ...aim of this study was to assess the antibiofilm activity of phages and develop phage cocktail to combat biofilm of P. mirabilis strains.
Methods and Results
Planktonic forms and biofilms of 50 tested uropathogenic P. mirabilis strains showed different sensitivity to 13 phages used. Phages 39APmC32, 65APm2833 and 72APm5211 presenting strong antibiofilm activity were selected as cocktail components. The antibiofilm activity of phage cocktails was similar or slightly higher than that of the most active phage. A three‐phage cocktail inhibited biofilm formation and destroyed biofilms of the same number of strains or 2–3 strains more compared to a single phage. The components of the three‐phage cocktail did not block each other's activity.
Conclusions
The potential of developed anti‐P. mirabilis phage cocktail as an antibiofilm agent was proved.
Significance and Impact of the Study
In this study, three broad host range phages presenting strong anti‐P. mirabilis biofilm activity were selected. Additionally, high stability of these viruses makes them a useful tool for controlling the biofilms.
Salmonella are important pathogenic bacteria and, following Campylobacter, they are the second most common cause of bacterial foodborne infections worldwide. To reduce the presence of bacteria along ...the food chain, the application of bacteriophages (phages) may be a promising tool. In this study, the lytic properties of six phages against five relevant Salmonella serotypes (S. Enteritidis, S. Typhimurium, S. Infantis, S. Paratyphi B and S. Indiana) were analyzed. Three phages were able to lyse all five serotypes. We determined the lytic potential of each phage on indicator strains in vitro at room temperature (RT) and at 37 ? using low multiplicities of infection (MOIs). Most phages reduced their host more efficiently at RT than at 37 ?, even at the lowest MOI of 0.001. Following this, the lytic activity of a cocktail comprising five phages (MOI = 0.1) was examined with each of the five serotypes and a mix of them at RT, 15, 12, 10, 8 and 6 ?. All cultures of single serotypes as well as the mixture of strains were significantly reduced at temperatures as low as 8 ?. For single serotypes, reductions of up to 5 logsub.10 units and up to 2.3 logsub.10 units were determined after 6 h (RT) and 40 h (8 ?), respectively. The mixture of strains was reduced by 1.7 logsub.10 units at 8 ?. The data clearly suggest that these phages are suitable candidates for biocontrol of various Salmonella serotypes under food manufacturing conditions.
A-1(L) is a freshwater cyanophage with a contractile tail that specifically infects
sp. PCC 7120, one of the model strains for molecular studies of cyanobacteria. Although isolated for half a ...century, its structure remains unknown, which limits our understanding on the interplay between A-1(L) and its host. Here we report the 3.35 Å cryo-EM structure of A-1(L) capsid, representing the first near-atomic resolution structure of a phage capsid with a T number of 9. The major capsid gp4 proteins assemble into 91 capsomers, including 80 hexons: 20 at the center of the facet and 60 at the facet edge, in addition to 11 identical pentons. These capsomers further assemble into the icosahedral capsid, via gradually increasing curvatures. Different from the previously reported capsids of known-structure, A-1(L) adopts a noncovalent chainmail structure of capsid stabilized by two kinds of mortise-and-tenon inter-capsomer interactions: a three-layered interface at the pseudo 3-fold axis combined with the complementarity in shape and electrostatic potential around the 2-fold axis. This unique capsomer construction enables A-1(L) to possess a rigid capsid, which is solely composed of the major capsid proteins with an HK97 fold.
Cyanobacteria are the most abundant photosynthetic bacteria, contributing significantly to the biomass production, O
generation, and CO
consumption on our planet. Their community structure and homeostasis in natural aquatic ecosystems are largely regulated by the corresponding cyanophages. In this study, we solved the structure of cyanophage A-1(L) capsid at near-atomic resolution and revealed a unique capsid construction. This capsid structure provides the molecular details for better understanding the assembly of A-1(L), and a structural platform for future investigation and application of A-1(L) in combination with its host
sp. PCC 7120. As the first isolated freshwater cyanophage that infects the genetically tractable model cyanobacterium, A-1(L) should become an ideal template for the genetic engineering and synthetic biology studies.
Type VI secretion systems (T6SSs) are newly identified contractile nanomachines that translocate effector proteins across bacterial membranes. The Francisella pathogenicity island, required for ...bacterial phagosome escape, intracellular replication, and virulence, was presumed to encode a T6SS-like apparatus. Here, we experimentally confirm the identity of this T6SS and, by cryo electron microscopy (cryoEM), show the structure of its post-contraction sheath at 3.7 Å resolution. We demonstrate the assembly of this T6SS by IglA/IglB and secretion of its putative effector proteins in response to environmental stimuli. The sheath has a quaternary structure with handedness opposite that of contracted sheath of T4 phage tail and is organized in an interlaced two-dimensional array by means of β sheet augmentation. By structure-based mutagenesis, we show that this interlacing is essential to secretion, phagosomal escape, and intracellular replication. Our atomic model of the T6SS will facilitate design of drugs targeting this highly prevalent secretion apparatus.
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•Specific environmental stimuli trigger Francisella T6SS assembly and secretion•Atomic structure of T6SS sheath provides targets for mutagenesis and drug design•Two-dimensional interlacing of T6SS sheath proteins is essential to its function•Lack of ClpV and different sheath structure support an alternative functional state
An atomic structure of the Francisella novicida type VI secretion system sheath, determined by cryoelectron microscopy, reveals a mesh-like architecture that is required for functional translocation of secreted proteins.
Performing fluorescence microscopy and electron microscopy on the same sample allows fluorescent signals to be used to identify and locate features of interest for subsequent imaging by electron ...microscopy. To carry out such correlative microscopy on vitrified samples appropriate for structural cryo-electron microscopy it is necessary to perform fluorescence microscopy at liquid-nitrogen temperatures. Here we describe an adaptation of a cryo-light microscopy stage to permit use of high-numerical aperture objectives. This allows high-sensitivity and high-resolution fluorescence microscopy of vitrified samples. We describe and apply a correlative cryo-fluorescence and cryo-electron microscopy workflow together with a fiducial bead-based image correlation procedure. This procedure allows us to locate fluorescent bacteriophages in cryo-electron microscopy images with an accuracy on the order of 50nm, based on their fluorescent signal. It will allow the user to precisely and unambiguously identify and locate objects and events for subsequent high-resolution structural study, based on fluorescent signals.
•Workflow for correlated cryo-fluorescence and cryo-electron microscopy.•Cryo-fluorescence microscopy setup incorporating a high numerical aperture objective.•Fluorescent signals located in cryo-electron micrographs with 50nm spatial precision.
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