Mycobacteriumphage Butters-encoded proteins contribute to host defense against viral attack

Abstract

25A diverse set of prophage-mediated mechanisms protecting bacterial hosts from 26 infection has been recently uncovered within Cluster N mycobacteriophages. In that 27 context, we unveil a novel defense mechanism in Cluster N prophage Butters. By using 28 bioinformatics analyses, phage plating efficiency experiments, microscopy, and 29 immunoprecipitation assays, we show that Butters genes located in the central region of 30 the genome play a key role in the defense against heterotypic viral attack. Our study 31 suggests that a two component system articulated by interactions between protein 32 products of genes 30 and 31 confers defense against heterotypic phage infection by 33 PurpleHaze or Alma, but is insufficient to confer defense against attack by the 34 heterotypic phage Island3. Therefore, based on heterotypic phage plating efficiencies 35 on the Butters lysogen, additional prophage genes required for defense are implicated. 36 37 IMPORTANCE 38 Many sequenced bacterial genomes including pathogenic bacteria contain prophages. 39 Some prophages encode defense systems that protect their bacterial host against 40 heterotypic viral attack. Understanding the mechanisms undergirding these defense 41 systems will be critical to development of phage therapy that circumvents these 42 defenses. Additionally, such knowledge will help engineer phage-resistant bacteria of 43 industrial importance.44 45 47they are useful in diagnostics of mycobacterial infections (1), the most notable of which 48 is tuberculosis (TB), and additionally can serve as genetic tools for mycobacteria (2-5). 49Most recently, engineered mycobacteriophages have been used in therapeutic 50 3 applications to combat infections from antibiotic-resistant strains of Mycobacterium 51 abscessus (6). To date over 11,000 mycobacteriophages have been isolated, over 52 1,800 sequenced, and over 1,600 are available in GenBank (7, 8). Mycobacteriophages 53 are a small subset of the estimated 10 31 bacteriophages existing in the biosphere (9). 54 Mycobacteriophages display high levels of genetic diversity and have been divided into 55 29 genomically similar clusters (A-AC) and a group of singletons with no close relatives 56 (10, 7). Although an increase in isolation and genomic characterization of 57 mycobacteriophages has occurred recently, the void in knowledge about gene 58 expression and function of mycobacteriophage gene products remains. 59 Prophages make up a majority of the known bacteriophage population (11). The 60 relationship between prophages and bacterial strains has shown numerous benefits to 61 both the hosts and phages. Prophages confer many advantages to the host upon 62 integration such as enhanced fitness, reduction of mutation rates, selective advantages, 63 and defense against additional viral attack (12). The bacterial host in turn provides the 64 phages with protection from harsh environments (12). In this context, numerous 65 mechanisms of defense have been recently discovered for Pseudomonas, 66 Mycobacterium, and Gordonia prophages...

Characterization of Novel Recombinant Mycobacteriophages derived from Homologous Recombination between two Temperate Phages

“…Additionally, Butters blocks infection by the heterotypic phage Island3 through a repressor-independent system (Dedrick et al . 2017; Mageeney et al . 2020).…”

“…Its repressor blocks infection by other cluster N phages (Dedrick et al 2017). Additionally, Butters blocks infection by the heterotypic phage Island3 through a repressorindependent system (Dedrick et al 2017;Mageeney et al 2020). To explore the mechanism of defense and to identify putative Island3 targets of Butters prophage defense, we pursued isolation of Island3 defense escape mutants (DEMs) -mutants that have escaped Butters prophage-mediated defense and consequently, can efficiently infect a Butters lysogen [mc 2 155(Butters)].…”

“…Cluster N mycobacteriophages, in particular, have been highlighted for the diversity that characterizes the central region of their genomes between more conserved sequences in the left and right arms (Dedrick et al 2017). Genes within the central region of cluster N mycobacteriophages are expressed in cluster N lysogens and have been implicated in prophage-mediated defense mechanisms that defend the host from heterotypic viral attack (Dedrick et al 2017;Mageeney et al 2020). Cluster N and I1 mycobacteriophage genomes are notable in their continuum of genetic similarity and are proposed to exchange genetic modules at a relatively faster rate (Mavrich and Hatfull 2017).…”

“…Genes within the central region of cluster N mycobacteriophages are expressed in cluster N lysogens and have been implicated in prophage-mediated defense mechanisms that defend the host from heterotypic viral attack (Dedrick et al . 2017; Mageeney et al . 2020).…”

Identification of a new antiphage system inMycobacteriumphage Butters

“…No reuse allowed without permission. smegmatis mc 2 155 lysogen against several heterotypic phages (26,28). Butters has a genome size of 41,491bp, a GC content of 66%, and an overall architecture similar to other cluster N phages (29).…”