The Protein Interactome of Mycobacteriophage Giles Predicts Functions for Unknown Proteins

Abstract

Mycobacteriophages are viruses that infect mycobacterial hosts and are prevalent in the environment. Nearly 700 mycobacteriophage genomes have been completely sequenced, revealing considerable diversity and genetic novelty. Here, we have determined the protein complement of mycobacteriophage Giles by mass spectrometry and mapped its genome-wide protein interactome to help elucidate the roles of its 77 predicted proteins, 50% of which have no known function. About 22,000 individual yeast twohybrid (Y2H) tests with four different Y2H vectors, followed by filtering and retest screens, resulted in 324 reproducible proteinprotein interactions, including 171 (136 nonredundant) high-confidence interactions. The complete set of high-confidence interactions among Giles proteins reveals new mechanistic details and predicts functions for unknown proteins. The Giles interactome is the first for any mycobacteriophage and one of just five known phage interactomes so far. Our results will help in understanding mycobacteriophage biology and aid in development of new genetic and therapeutic tools to understand Mycobacterium tuberculosis. IMPORTANCEMycobacterium tuberculosis causes over 9 million new cases of tuberculosis each year. Mycobacteriophages, viruses of mycobacterial hosts, hold considerable potential to understand phage diversity, evolution, and mycobacterial biology, aiding in the development of therapeutic tools to control mycobacterial infections. The mycobacteriophage Giles protein-protein interaction network allows us to predict functions for unknown proteins and shed light on major biological processes in phage biology. For example, Giles gp76, a protein of unknown function, is found to associate with phage packaging and maturation. The functions of mycobacteriophage-derived proteins may suggest novel therapeutic approaches for tuberculosis. Our ORFeome clone set of Giles proteins and the interactome data will be useful resources for phage interactomics.T he continuous emergence of bacterial pathogens resistant to antibiotics is an increasing medical problem (1). Mycobacterium tuberculosis is prominent among these pathogens, with over 9 million new cases of tuberculosis reported each year. There is an urgent need for alternate ways to control M. tuberculosis infections, and one potential strategy involves using mycobacteriophages for prophylaxis or therapy (2). The emergence of extensively drug-resistant (XDR) and totally drug-resistant (TDR) strains of M. tuberculosis, both of which are especially difficult to control (3), has spurred renewed interest in the therapeutic use of bacteriophages.Mycobacteriophages are known to infect many different species of both fast-and slow-growing mycobacteria, including M. tuberculosis and Mycobacterium smegmatis (4, 5). Over the past decade, thousands of mycobacteriophages have been isolated, and hundreds have been completely sequenced (http://phagesdb.org/). Mycobacteriophage genomes are highly mosaic due to horizontal genetic exchange (6-8) but can be grouped into 2...

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“…The highest number of positive results was obtained with the proteins expressed from the combination of the vectors pGBGT7g and pGADT7g (fused at their N termini [NN]), followed by pGBGT7g and pGADCg (fused at the N terminus and C terminus, respectively [NC]), pGBKCg and pGADT7g (fused at their C terminus and N terminus, respectively [CN]), and pGBKCg and pGADCg (fused at their C termini [CC]) (Table S6; Fig. 3 and 4), which is in line with the results previously obtained for other bacteriophages (45,46).…”

“…This reporter allows the background due to the autoactivation of the proteins under study to be reduced through the addition of 3-amino-1,2,4-triazole (3AT), a competitive inhibitor of the His3 protein (see Materials and Methods) (43). This approach has been applied successfully to the study of phage interactomes from different hosts, including Escherichia coli bacteriophage , Streptococcus phage Cp-1, or Mycobacterium phage Giles (45)(46)(47).…”

Mycobacteriophages

“…The number of uncharacterized genes is vast, but the methods are available for their genetic, biochemical, and structural characterization, and the limiting factor may be the development of suitable assays. Surrogate approaches such as screening for toxicity (62, 106) and finding interacting partners (136, 137) should be useful strategies. Because many of the phage genes may play roles in microbial dynamics, the availability of such a large collection offers a very substantial resource.…”

Mycobacteriophage Fruitloop gp52 inactivates Wag31 (DivIVA) to prevent heterotypic superinfection

“…Elucidating gene functions is facilitated by constructing mutant phage derivatives in which specific phage genes have been deleted (Marinelli et al, 2008), although many genes are not required for lytic growth and do not exhibit informative phenotypes (Pope et al, 2011;Dedrick et al, 2013). Other strategies to characterize gene functions include determining the patterns of interacting phage proteins (Blasche et al, 2013, Mehla et al, 2015Mariano et al, 2016) and identification of phageencoded genes that are toxic to the bacterial host and identifying interacting host proteins (Liu et al, 2004). This also has the potential to identify essential host genes and their exploitation as targets for antibiotic development (Liu et al, 2004).…”