Single Particle Asymmetric Reconstructions of Stx Bacteriophage Phi24B

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Human gut microbiome comprises a wide variety of organisms, from fungi to bacteria. Escherichia coli is the successor of such organisms in case of completeness of global research, from ecology to genomics. Additionally, it is well known that a range of E. coli strains can cause infections. For example, Shiga toxin-producing E. coli (STEC), known as dangerous food-born pathogen, inducing severe hemorrhagic colitis. This line gains its pathogenic phenotype as a result of infection by Shiga toxin-encoding phages (Stx phages). Notably, the toxin is produced only during the lytic cycle of the virus. Although the global incidence of STEC infection is low, severe disease is observed in about 10% of affected individuals with a mortality rate of 3–5%. The toxin production depends on the in situ induction level in the pathogen’s population developed in a patient. Therefore, the usage of antibiotics increasing the prophage induction is not recommended for the STEC infection treatment [1]. The Stx bacteriophage vB_EcoP-24B (hereafter referred to as phi24B) was initially isolated from a clinical STEC strain of the O157:H7 serotype. The phi24B related phages were later found in more than half of STEC genomes. The genome and physiology of phi24B-related phages were extensively studied [2]. In recent years a wide variety of tailed phages was studied using structural methods, particularly with Cryo-EM. Structural data is indispensable in areas of phage physiology. However, the structural organization of phi24B virion and other Stx phages remains poorly understood. In our study we present new insights in Tail-Capsid connection and genome remnants after ejection. Cryo-EM of purified phi24B sample was performed with Titan Krios 300 kV microscope equipped with a Gatan K3 camera. Samples were applied to the glow discharged Quantifoil 1.2/1.3 holey carbon support film and imaged with pixel size 0.83Å, dose of 53e-/Å2 and -2.5 micrometer defocus. Data processing was carried out with cryoSPARC software [3]. The Single Particle Analysis pipeline involved template-based particle picking for the capsid particles. As a result, the total numbers of collected individual projections of empty and filled with DNA particles are 18120 and 18400, which is a sustainable clean dataset for asymmetric whole virion reconstruction. Based on GP4 phage asymmetric reconstruction [4], phi24B whole virion densities were obtained. Next, 3D variability cluster analysis with focus on tail region was conducted to resolve smeared tail density. As a result, in both ejection states two tail positions relative to the capsid were observed. In Empty (upon spontaneous DNA ejection into the solution) state there is a turn along the tail axis of six degrees between two states. In the Filled dataset two discrete states of the same turn with tilt of the tail relative to the capsid by two degrees were found. For both states were collected 9656 and 8748 particles for Filled phages and 7716 and 10407 particles for Emptied virions. The resolution numbers for two states are 5.6 and 5.7 Å for Filled phages, 6.3 and 5.8 Å for Emptied virions. Such notable tilt in Filled phi24B virion is likely to be as a result of inner genome pressure on capsid walls and portal. At the same time, in Empty dataset the tail is discovered to be aligned along the central axis. These observations allow to suggest that a significant inner pressure likely makes the tail deviate from perfect position in Filled phages. The feature found in phi24B was not previously pointed out in previous literature on phages. In Empty virions a presence of circular cables covering inner capsid surface was clearly observed. There are 16 such rings on different head levels, from portal to cap. Therefore, we hypothesize these rings to be DNA remnants after ejection. The estimated length of the remaining DNA is about 6-7 Kbp. We speculate that in case of infection when the phage genome gets internalized by the host cell, the additional force driving the DNA into the cell is produced by complex physico-chemical effect [5]. This force helps to detach the DNA contacting with the capsid wall. In the case of spontaneous ejection into the solution the fragment of the DNA remains stuck inside the virion and the rest of the molecule gets removed by the background nuclease activity present in the medium [6]. CryoEM reconstructions of phi24B virion in two states. Comparison of Emptied and Filled states (B), A and C – GSFSC plots of tail turn pairs of Empty (A) and Filled (B) states, D and G – side views of overlapped turn pairs of Emptied (D) and Filled (G) phi24B reconstructions, E and F – same volumes with tails pointed to viewer.

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