Functional characterization of a bacteriophage-encoded inhibitor of Staphylococcus aureus transcription

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No description available.

No description available.

No description available.

Throughout recent history scientists have struggled to elucidate the biochemical and biophysical mechanisms that guide the assembly of macromolecular structures. The early models of "sub-assembly" or "self assembly" attempted to explain how individual components could interact in a precisely regulated manner to form higher-ordered complex biological structures. Subsequent studies, using viral systems as assembly models, demonstrated how protein-protein and protein-nucleic acid interactions assist in lowering the thermodynamic barriers that typically disfavor assembly.Due to their simplicity, viruses provide an ideal system to investigate the biophysical mechanisms that drive the assembly of complex biological structures. Proper virion assembly requires numerous macromolecular interactions that proceed along an ordered morphogenetic pathway. While structural proteins are incorporated into the final product, morphogenesis is equally dependent upon scaffolding proteins, which are not included in the mature virion. Since the identification of scaffolding proteins in the bacteriophage P22, homologues have been discovered in many systems. Scaffolding proteins play multiple roles during morphogenesis by inducing protein conformational switches and lowering the thermodynamic barriers to promote virion assembly, while ensuring the efficiency and fidelity of the final product.

The purpose of this research was to explore the potential application of lytic phages to control Cronobacter spp. in infant formula. More than two hundred and fifty phages were isolated from various environmental samples against different strains of Cronobacter spp. Selected phages were characterized by morphology, host range, and cross infectivity. The genomes of five novel Cronobacter phages [vB_CsaM_GAP31 (GAP31), vB_CsaM_GAP32 (GAP32), vB_CsaP_GAP52 (GAP52), vB_CsaM_GAP161 (GAP161), vB_CsaP_GAP227 (GAP227)] were sequenced. Phage GAP32 possess the second largest phage genome sequenced to date, and it is proposed that GAP32 belongs to a new genus of “Gap32likeviruses”. Phages GAP52 and GAP227 are the first C. sakazakii podoviruses whose genomes have been sequenced. None of the sequenced genomes showed homology to virulent or lysogenic genes. In addition, in vivo administration of phage GAP161 in the hemolymph of Galleria mellonella larvae showed no negative effects on the wellbeing of the larvae and could effectively prevent Cronobacter infection in the larvae. A cocktail of five phages was highly effective for biocontrol of three Cronobacter sakazakii strains present as a mixed culture in both broth media and contaminated reconstituted infant formula. This phage cocktail could be potentially used to control C. sakazakii during preparation of infant formula but would first have to be clinically evaluated in mammalian models. / NSERC & DFO

The growing threat of drug- resistant Staphylococcus aureus (S. aureus) infections mandates the need to develop novel, effective and alternative antibacterial therapeutics. Despite infection prevention and control measures, methicillin resistant S. aureus (MRSA)-associated deaths reached 11,285 in 2011 in the USA (CDC, 2013). To counteract the threat of drug resistant S. aureus, we sought to construct and characterize a novel therapeutic based on the display of lytic antibacterial enzymes, termed endolysins. These endolysins were displayed on the surface of a specific bacterial virus, bacteriophage (phage), to generate lytic antibacterial nanoparticles. Endolysins are encoded individually by a variety of double-stranded DNA phage and act to direct host lysis and escape. These lytic enzymes confer a high degree of host specificity that could potentially substitute for, or be combined with, antibiotics in the treatment of gram-positive drug resistant bacterial infections such as MRSA. In this study, modular domains of the phage-encoded endolysin K enzyme, specific to S. aureus, were displayed on the capsid surface of phage lambda () via fusion with the λ major head (capsid) protein, gpD. The constructs of displayed endolysins were prepared in various combinations to maximize the functional display of gpD::X fusions on the phage. Phage lysates were generated, collected and purified and lysis was investigated by adding to fresh lawns of MRSA, vancomycin resistant S. aureus (VRSA) and bovine S. aureus. Phage preparations did not readily confer cell lysis, likely due to poor incorporation of the fusions onto the functional phage capsid. We purified the fusion proteins (gpD::X) and tested them for their lytic activity. We noted that the activity of the gpD::LysK protein was not impaired by the fusion and demonstrated lysis on live and dead (autoclaved) bovine S. aureus. In contrast to gpD::LysK, the gpD::CHAP protein fusion, expressing only the CHAP catalytic domain of endolysin K showed variable results in the lysis assays that we performed. In the zymogram assay, gpD::CHAP did not elicit any observable lysis on live bovine S. aureus cells, but did effectively lyse dead cells of the same S. aureus species; however, it was highly lytic in the inhibition assay on bovine S. aureus. The CHAP::gpD protein fusion, which is the CHAP domain fused to the N terminus of gpD only showed its ability to inhibit bovine S. aureus growth on the inhibition assay. The fusion of endolysin K or its CHAP domain to gpD protein does not seem to interfere with lytic activity, but may result in recalcitrant gpD fusions that compromise the ability to efficiently decorate the phage capsid. Suggestions for improved fusion capsid integration are discussed.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2001. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 128-138). Available also in a digital version from Dissertation Abstracts.