Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa

Artilysins constitute a novel class of efficient enzyme-based antibacterials. Specifically, they covalently combine a bacteriophage-encoded endolysin, which degrades the peptidoglycan, with a targeting peptide that transports the endolysin through the outer membrane of Gram-negative bacteria. Art-085, as well as Art-175, its optimized homolog with increased thermostability, are each composed of the sheep myeloid 29-amino acid (SMAP-29) peptide fused to the KZ144 endolysin. In contrast to KZ144, Art-085 and Art-175 pass the outer membrane and kill Pseudomonas aeruginosa, including multidrug-resistant strains, in a rapid and efficient (similar to 5 log units) manner. Time-lapse microscopy confirms that Art-175 punctures the peptidoglycan layer within 1 min, inducing a bulging membrane and complete lysis. Art-175 is highly refractory to resistance development by naturally occurring mutations. In addition, the resistance mechanisms against 21 therapeutically used antibiotics do not show cross-resistance to Art-175. Since Art-175 does not require an active metabolism for its activity, it has a superior bactericidal effect against P. aeruginosa persisters (up to > 4 log units compared to that of the untreated controls). In summary, Art-175 is a novel antibacterial that is well suited for a broad range of applications in hygiene and veterinary and human medicine, with a unique potential to target persister-driven chronic infections

Bacteria-Killing Type IV Secretion Systems

Bacteria have been constantly competing for nutrients and space for billions of years. During this time, they have evolved many different molecular mechanisms by which to secrete proteinaceous effectors in order to manipulate and often kill rival bacterial and eukaryotic cells. These processes often employ large multimeric transmembrane nanomachines that have been classified as types I–IX secretion systems. One of the most evolutionarily versatile are the Type IV secretion systems (T4SSs), which have been shown to be able to secrete macromolecules directly into both eukaryotic and prokaryotic cells. Until recently, examples of T4SS-mediated macromolecule transfer from one bacterium to another was restricted to protein-DNA complexes during bacterial conjugation. This view changed when it was shown by our group that many Xanthomonas species carry a T4SS that is specialized to transfer toxic bacterial effectors into rival bacterial cells, resulting in cell death. This review will focus on this special subtype of T4SS by describing its distinguishing features, similar systems in other proteobacterial genomes, and the nature of the effectors secreted by these systems and their cognate inhibitors

Impact of phage-host interactions on the behavior of Salmonella Typhimurium

Salmonella enterica strains significantly contribute to the infectious disease burden in both developed and developing countries, with a majority of their infections being food- or waterborne. Since the evolution and physiology of these important pathogens are partly shaped by the predatory, parasitic and symbiotic interactions with the bacterial viruses (i.e. bacteriophages or phages) that tend to infect them, this dissertation has focused on dissecting the impact of phage P22 infection on S. Typhimurium behavior. While population-level approaches have traditionally described phage host interactions of temperate phages such as P22, as being either lytic or lysogenic, it was decided to more closely examine P22 S. Typhimurium infection dynamics at the single cell level, by using a fluorescent DNA labelling approach that enabled specific tracking of the whereabouts of the P22 chromosome during all stages of infection using time-lapse fluorescent microscopy (chapter 3). Aside from revealing interesting dynamics of phage and host DNA, this analysis also provided molecular evidence of the existence of the phage carrier state in which an episomal cluster of P22 genomes remains located at the cell pole of the host cell and becomes segregated asymmetrically between siblings. It seems that this state invariably precedes either lytic or lysogenic proliferation. Moreover, during establishment of lysogeny, phage free cells were able to segregate from the phage carrier cell before actual phage chromosome integration occurred, and experiments further revealed that these phage free siblings remained resistant to superinfection by P22 during several generations, suggesting the cytoplasmic inheritance of a resistance factor. This transient resistance is hypothesized to increase the window of stable coexistence between phages and bacteria (chapter 3). The P22 pid (P22 encoded instigator of dgo expression) gene was previously discovered in our research group and found to specifically derepress the dgoRKDAT operon (encoding the necessary proteins for galactonate metabolism) in S. Typhimurium. Further investigation of the pid gene and Pid protein in this study now revealed that its expression was tightly linked to the phage carrier state of phage P22 (chapter 4). Although protein-protein interaction studies so far have failed to identify interaction partners of the host, they could demonstrate that Pid interacts with itself. In fact, the first crystal structure model of Pid revealed it to form a trimeric structure, due to a coiled-coil formation from the alfa-helices of three Pid molecules. The exact molecular mechanism by which Pid mediates dgoRKDAT derepression, as well as the physiological impact of the Pid/dgoRKDAT interaction for S. Typhimurium remain subject to further study. In essence, using dynamic (single) cell biology this dissertation has uncovered novel phage host interactions between S. Typhimurium and P22 that surpass the classical bifurcation of lytic and lysogenic phage development. More specifically, by closely examining the phage carrier state, it was shown that this state is able to specifically affect gene expression of the host cell, and that it allows for the emergence of phage free siblings that are transiently resistant to P22 infection.nrpages: 138status: publishe

P22 mediated recombination of frt-sites

Flp mediated site specific recombination of frt-sites is frequently used in genetic engineering to excise, insert or invert DNA-cassettes in the chromosome. While constructs flanked by frt-sites are generally considered to be stable in the absence of the Flp enzyme, we observed that P22 chromosomes exceeding wild-type length tend to lose frt-flanked insertions via Flp independent recombination of frt-sites during phage propagation. This spontaneous recombination should be considered when engineering the chromosome of P22 and perhaps of other phages as well.publisher: Elsevier articletitle: P22 mediated recombination of frt-sites journaltitle: Virology articlelink: http://dx.doi.org/10.1016/j.virol.2014.06.015 content_type: article copyright: Copyright © 2014 Elsevier Inc. All rights reserved.status: publishe

Viral Transmission Dynamics at Single-Cell Resolution Reveal Transiently Immune Subpopulations Caused by a Carrier State Association

<div><p>Monitoring the complex transmission dynamics of a bacterial virus (temperate phage P22) throughout a population of its host (<i>Salmonella</i> Typhimurium) at single cell resolution revealed the unexpected existence of a transiently immune subpopulation of host cells that emerged from peculiarities preceding the process of lysogenization. More specifically, an infection event ultimately leading to a lysogen first yielded a phage carrier cell harboring a polarly tethered P22 episome. Upon subsequent division, the daughter cell inheriting this episome became lysogenized by an integration event yielding a prophage, while the other daughter cell became P22-free. However, since the phage carrier cell was shown to overproduce immunity factors that are cytoplasmically inherited by the P22-free daughter cell and further passed down to its siblings, a transiently resistant subpopulation was generated that upon dilution of these immunity factors again became susceptible to P22 infection. The iterative emergence and infection of transiently resistant subpopulations suggests a new bet-hedging strategy by which viruses could manage to sustain both vertical and horizontal transmission routes throughout an infected population without compromising a stable co-existence with their host.</p></div

Categorizing single cell infection dynamics in the P22–<i>S</i>. Typhimurium model system.

<p>In the LT2ΔpSLT/pALA2705 reporter, presence of a P22 chromosome is revealed by the formation of a fluorescent GFP focus originating from GFP-ParB molecules bound to the <i>parS</i> sequence engineered in P22 <i>parS</i>. (A) The central picture shows an initial snapshot of an exponential phase population of LT2ΔpSLT/pALA2705 infected with P22 <i>parS</i> (MOI = 20) and further grown for 4 hours post infection in a semi-continuous culture. Time-lapse recordings of specific events are presented as a zoom in on the original snapshot as indicated by the black lines (box A1–A4). Four types of phage–host associations are seen in panel A: lysogenized cells in which the stably integrated P22 <i>parS</i> prophage yields a discrete GFP focus that replicates and segregates together with the host chromosome (box A1); lytically infected cells in which the replicating P22 <i>parS</i> chromosome yields a more diffuse and randomly dispersed GFP cloud throughout the cell prior to cell lysis (box A2); P22-free cells in which the absence of a P22 <i>parS</i> chromosome yields a diffuse cytoplasmic GFP fluorescence (box A3); phage carrier cells in which a polarly tethered P22 <i>parS</i> episome yields a coherent GFP cloud in one of the cell poles (box A4). Please note that the bright fluorescent cell at the bottom of panel A is a rare artifact. (B-D) Time-lapse series of (B) cells in the absence of P22 <i>parS</i>, (C) of growing cells from a P22 <i>parS</i> lysogen in LT2ΔpSLT/pALA2705, and (D) of LT2ΔpSLT/pALA2705 cells infected with P22 <i>c2 parS</i> (an obligate lytic derivative of P22 <i>parS</i>). (E) Snapshots from the lineages emerging from two phage carrier cells within a P22 <i>parS</i> infected LT2ΔpSLT/pALA2705 population, exhibiting either direct (left panel) or delayed (right panel) integration of the P22 <i>parS</i> prophage, resulting either in a homogeneous population of lysogens (left panel) or a heterogeneous population of both lysogens and P22-free cells (right panel). Analysis of 114 such lineages revealed the segregation of P22-free siblings in ca. 41% of cases. Phase contrast images (showing the cells) and GFP signal (reporting the P22 <i>parS</i> chromosomes) are merged. A 5 μm scale bar is shown at the bottom right of each panel. Timestamps are shown in the top left corners of time-lapse images. In panel D the timestamp is set at 0 min from the moment a ParB-GFP foci became visible. In all other panels the timestamp was started when first image was taken.</p

P22 infection can endow P22 immunity upon a subpopulation of P22-free host cells.

<p>(A) An exponential phase LT2ΔpSLT/pALA2705 population infected with P22 <i>parS</i> (MOI = 20) was grown for 4 hours in a semi-continuous culture after which an aliquot was mixed with P22 H5 (an obligate lytic mutant of P22; MOI = 20) and subjected to time-lapse microscopy. Arrows indicate phage free cells in the population (as recognized by their diffuse GFP-ParB fluorescence) remaining resistant to P22 H5 and P22 <i>parS</i> infection for over 300 minutes (left panel). Out of the 383 cells observed in this experiment, ca. 20% appeared to be P22-free and resistant. (B) An LT2ΔpSLT/pALA2705 control population grown in the same manner as in (A) but not previously exposed to P22 <i>parS</i>, either lysed or ceased growth when exposed to P22 H5 (MOI = 20). Phase contrast images (showing the cells) and GFP signal (reporting the P22 <i>parS</i> chromosomes) are merged. A 10 μm scale bar is shown at the bottom right of each panel. Timestamp in the upper left corner indicates time after mixing with P22 H5.</p