Chapter 5 - Genomics of Staphylococcal Twort-like Phages - Potential Therapeutics of the Post-Antibiotic Era

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Abstract

Polyvalent bacteriophages of the genus Twort-like that infect clinically relevant Staphylococcus strains may be among the most promising phages with potential therapeutic applications. They are obligatorily lytic, infect the majority of Staphylococcus strains in clinical strain collections, propagate efficiently and do not transfer foreign DNA by transduction. Comparative genomic analysis of 11 S. aureus/S. epidermidis Twort-like phages, as presented in this chapter, emphasizes their strikingly high similarity and clear divergence from phage Twort of the same genus, which might have evolved in hosts of a different species group. Genetically, these phages form a relatively isolated group, which minimizes the risk of acquiring potentially harmful genes. The order of genes in core parts of their 127 to 140-kb genomes is conserved and resembles that found in related representatives of the Spounavirinae subfamily of myoviruses. Functions of certain conserved genes can be predicted based on their homology to prototypical genes of model spounavirus SPO1. Deletions in the genomes of certain phages mark genes that are dispensable for phage development. Nearly half of the genes of these phages have no known homologues. Unique genes are mostly located near termini of the virion DNA molecule and are expressed early in phage development as implied by analysis of their potential transcriptional signals. Thus, many of them are likely to play a role in host takeover. Single genes encode homologues of bacterial virulence‐associated proteins. They were apparently acquired by a common ancestor of these phages by horizontal gene transfer but presumably evolved towards gaining functions that increase phage infectivity for bacteria or facilitate mature phage release. Major differences between the genomes of S. aureus/S. epidermidis Twort-like phages consist of single nucleotide polymorphisms and insertions/deletions of short stretches of nucleotides, single genes, or introns of group I. Although the number and location of introns may vary between particular phages, intron shuffling is unlikely to be a major factor responsible for specificity differences.

Introduction

The Staphylococcus genus groups several species of Gram-positive bacteria that inhabit human or animal organisms. Many of these species, especially Staphylococcus aureus, include highly pathogenic strains that can cause infections, which are manifested by various symptoms ranging from relatively mild to life threatening (reviewed by Tenover and Gorwitz, 2006). The treatment of such infections is becoming increasingly problematic due to the dissemination of methicillin-resistant S. aureus, as well as the emergence of vancomycin-resistant strains (Arias and Murray, 2009, Chambers and DeLeo, 2009, Howden et al., 2010). Naturally occurring, virulent bacteriophages that infect and kill a wide range of staphylococcal strains may become an alternative in the treatment of otherwise incurable infections caused by antibiotic-resistant staphylococci (Borysowski, 2011, Mann, 2008, Merabishvili et al., 2009). A few of them were shown to be effective in the treatment of staphylococcal infections in animals or are used in phage therapy including clinical trials in humans (Capparelli et al., 2007, Gill et al., 2006; Gupta and Prasad, 2011, Jikia et al., 2005, Kvachadze et al., 2011, Markoishvili et al., 2002, Merabishvili et al., 2009, Paul et al., 2011a; O'Flaherty et al., 2005b, Rhoads et al., 2009; Sulakvelidze et al., 2001; Sulakvelidze and Kutter, 2005, Sunagar et al., 2010, Wills et al., 2005; Zimecki et al., 2008, Zimecki et al., 2009, Zimecki et al., 2010). Additionally, proteins of these phages that are lethal to Staphylococci may serve as prototypes of antibacterials, mark potential drug targets in staphylococcal cells, or can be used as anti-staphylococcal agents by themselves (Fischetti, 2008, Gu et al., 2011; Liu et al., 2004, Pastagia et al., 2011, Projan, 2004; Rashel et al., 2007). Phages of the most promising group from a therapeutic point of view belong to the Twort-like genus (Klumpp et al., 2010). Although their history dates as far back as the history of phages, with their first representative, phage Twort, believed to have been isolated in 1915 (Lavigne et al., 2009, Twort, 1915), little is yet known about the biology of these phages. This chapter provides the genome-wide comparison of 11 staphylococcal Twort-like phages, which are suitable for therapeutic applications. Some genomic features of these phages which appear to be responsible for their certain properties and wide strain specificity are also discussed.

Section snippets

Staphylococcal Bacteriophages: A Short Overview

Early interest in staphylococcal phages was stimulated by their potential therapeutic applications (reviewed by Alisky et al., 1998, Sulakvelidze and Kutter, 2005; Sulakvelidze et al., 2001). However, after the introduction of antibiotics, it was motivated mainly by the wide application of phages to differentiate clinical staphylococcal strains. The method of phage typing of staphylococci derived from this early work and originally developed by Wilson and Atkinson (1945) has been used,

Morphology

Morphologically, staphylococcal Twort-like phages resemble phages of the same genus that infect Listeria monocytogenes (A511, P100) and differ from Bacillus phage SPO1 by having longer tails (from 175–176 nm in ISP and 676Ż to 217–219 nm in K and P4W/Fi200W versus 140 nm in SPO1), which are also slightly more slender (Fig. 1; Duda et al., 2006, Hotchin, 1954, Jarvis et al., 1993; Klumpp et al., 2008, 2010, and references therein; Kwiatek et al., 2012; Ulatowska et al., in preparation). However,

Concluding Remarks

Genomic characterization of large obligatorily virulent bacteriophages on a mass scale became possible only recently with the advent of high-throughput cloning-independent sequencing technologies. The comparison of 11 phage genomic sequences presented in this chapter is only a small step toward understanding the biology of staphylococcal Twort-like phages and the biology of the Spounavirinae subfamily of myoviruses they belong to. Functional analysis of knockout mutants in genes of undetermined

Acknowledgments

The authors are grateful to Hans-Wolfgang Ackermann for help in the description of phage morphological features and to Wacław Szybalski for helpful comments on this manuscript. This work was supported by funds from the Operational Program ‘Innovative Economy, 2007-2013’ (Priority axis 1. Research and Development of Modern Technologies, Measure 1.3 Support for R&D projects for entrepreneurs carried out by scientific entities, Submeasure 1.3.1, Development Project No. POIG 01.03.01-02-003/08

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