Evgeniya V. Pechnikova

Genome packaging in EL and Lin68, two giant phiKZ-like bacteriophages of P. aeruginosa

A unique feature of the Pseudomonas aeruginosa giant phage phiKZ is its way of genome packaging onto a spool-like protein structure, the inner body. Until recently, no similar structures have been detected in other phages. We have studied DNA packaging in P. aeruginosa phages EL and Lin68 using cryo-electron microscopy and revealed the presence of inner bodies. The shape and positioning of the inner body and the density of the DNA packaging in EL are different from those found in phiKZ and Lin68. This internal organization explains how the shorter EL genome is packed into a large EL capsid, which has the same external dimensions as the capsids of phiKZ and Lin68. The similarity in the structural organization in EL and other phiKZ-like phages indicates that EL is phylogenetically related to other phiKZ-like phages, and, despite the lack of detectable DNA homology, EL, phiKZ, and Lin68 descend from a common ancestor.

TL, the new bacteriophage of Pseudomonas aeruginosa and its application for the search of halo-producing bacteriophages

The properties of new virulent bacteriophage TL of Pseudomonas aeruginosa belonging to the family Podoviridae (genome size of 46 kb) were investigated. This bacteriophage is capable of lysing the bacterial lawn in halo zones around negative colonies (NC) of other bacteriophages. TL forms large NC, that are hardly distinguishable on the lawn of P. aeruginisa PAO1. At the same time, on the lawns of some phage-resistant PAO1 mutants, as well as on those produced by a number of clinical isolates, TL forms more transparent NC. It is suggested that more effective growth of the bacteriophage TL NC is associated with the differences in outer lipopolysaccharide (LPS) layer of the cell walls of different bacterial strains, as well as of the bacteria inside and outside of the halos. This TL property was used to optimize selection of bacteriophages producing halos around NC on the lawn of P. aeruginosa PAO1. As a result, a group of bacteriophages differing in the patterns of interaction between their halos and TL bacteriophage, as well as in some characters was identified. Taking into consideration the importance of cell-surfaced structures of P. aeruginosa in manifestation of virulence and pathogenicity, possible utilization of specific phage enzymes, polysacchadide depolymerases, for more effective treatment of P. aeruginosa infections is discussed.

Structure and properties of virions and virus-like particles derived from the coat protein of Alternanthera mosaic virus

Plant viruses and their virus-like particles (VLPs) have a lot of advantages for biotechnological applications including complete safety for humans. Alternanthera mosaic virus (AltMV) is a potentially promising object for design of novel materials. The 3D structures of AltMV virions and its VLPs were obtained by single particle EM at ~13Å resolution. The comparison of the reconstructions and a trypsin treatment revealed that AltMV CPs possesses a different fold in the presence (virions) and absence of viral RNA (VLPs). For the first time, the structure of morphologically similar virions and virus-like particles based on the coat protein of a helical filamentous plant virus is shown to be different. Despite this, both AltMV virions and VLPs are stable in a wide range of conditions. To provide a large amount of AltMV for biotechnology usage the isolation procedure was modified.

Structure and Virulence of Pseudomonas aeruginosa Bacteriophages

We used the single-particle cryo-electron microscopy and three-dimensional (3D) structural approach to solve the 3D structures of several bacteriophages that infects Pseudomonas aeruginosa. These gram-negative bacteria are frequently pathogenic in humans, and can cause severe diseases such as nephropathy, fibrosis and inflammation. Therefore, they have high potential for phage therapy [1,2]. We calculated the 3D reconstructions of the capsids of the following bacteriophages: SN, KMV, YuA at the resolution 35Å and better (table 1). The capsids of all phages except YuA represent the regular icosahedrons with a triangulation T=9 (SN) and T=7 (KMV). YuA bacteriophage represents the elongated icosahedron with the C5 type of symmetry. All phages possess the unique surface architecture of their capsids with prominent décor elements (Fig. 1, A-C). Using the polyclonal antibodies directed against structural components: pg22 of the phage SN, pg47 of the phage KMV and pg63 of the phage YuA we were able to demonstrate the surface distribution of these structural proteins (Fig.2, A-C). To reveal the structural principles of phage virulence, we used the electron tomography as a powerful tool to study the interactions of phages with the wild type and mutant P. aeruginosa. The tomographic low-resolution reconstructions explained the specificity while binding of phages to bacteria cell surface for initiation of infection.