Molecular Genetics of Mycobacteriophages

Hatfull, Graham F.

doi:10.1128/microbiolspec.mgm2-0032-2013

Cited by 77 publications

(47 citation statements)

References 217 publications

(318 reference statements)

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“…In mycobacteria, shuttle phasmids (8) have been critical to the development of genetic manipulation systems for mycobacteria, including the development of transformation systems, transposon mutagenesis, recombination substitution mutagenesis, and transfer of point mutations (9-13), as reviewed extensively by Hatfull and Jacobs (14). Thus, TM4-and D29-based recombinant mycobacteriophages make attractive tools for mycobacterial research due to their broad host range, which includes many species of mycobacteria.…”

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Fluorescent Reporter DS6A Mycobacteriophages Reveal Unique Variations in Infectibility and Phage Production in Mycobacteria

et al. 2016

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Mycobacteriophage DS6A is unique among the more than 8,000 isolated mycobacteriophages due to its ability to form plaques exclusively on mycobacteria belonging to the Mycobacterium tuberculosis complex (MTBC). Speculation surrounding this specificity has led to unsupported assertions in published studies and patents that nontuberculous mycobacteria (NTM) are wholly resistant to DS6A infection. In this study, we identified two independent nonessential regions in the DS6A genome and replaced them with an mVenus-expressing plasmid to generate fluorescent reporter phages ⌽ 2 GFP12 and ⌽ 2 GFP13. We show that even though DS6A is able to form plaques only on MTBC bacteria, infection of various NTM results in mVenus expression in transduced cells. The efficiency of DS6A in delivering DNA varied between NTM species. Additionally, we saw a striking difference in the efficiency of DNA delivery between the closely related members of the Mycobacterium abscessus complex, M. abscessus and Mycobacterium massiliense. We also demonstrated that TM4 and DS6A, two phages that do not form plaques on M. massiliense, differ in their ability to deliver DNA, suggesting that there is a phage-specific restriction between mycobacterial species. Phylogenetic analysis reveals that the DS6A genome has a characteristically mosaic structure but provided few insights into the basis for the specificity for MTBC hosts. This study demonstrates that the inability of the MTBC-specific phage DS6A to form plaques on NTM is more complex than previously thought. Moreover, the DS6A-derived fluorophages provide important new tools for the study of mycobacterial biology. IMPORTANCEThe coevolution of bacteria and their infecting phages involves a constant arms race for bacteria to prevent phage infection and phage to overcome these preventions. Although a diverse array of phage defense systems is well characterized in bacteria, very few phage restriction systems are known in mycobacteria. The DS6A mycobacteriophage is unique in the mycobacterial world in that it forms plaques only on members of the Mycobacterium tuberculosis complex. However, the novel DS6A reporter phages developed in this work demonstrate that DS6A can infect nontuberculous mycobacteria at various efficiencies. By comparing the abilities of DS6A and another phage, TM4, to infect and form plaques on various mycobacterial species, we can begin to discern new phage restriction systems employed within the genus. The standard treatment for a drug-susceptible Mycobacterium tuberculosis infection is four drugs (rifampin, isoniazid, pyrazinamide, and ethambutol) for 2 months, followed by 4 months of two drugs (rifampin and isoniazid). One of the key aspects of successfully treating tuberculosis is ensuring that the infecting strain is susceptible to each of the antibiotics given; therefore, quickly diagnosing antibiotic resistance is important for successful patient outcome as well as for lowering the incidence of the generation of drug-resistant mutants from improper antibiotic administra...

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Fluorescent Reporter DS6A Mycobacteriophages Reveal Unique Variations in Infectibility and Phage Production in Mycobacteria

et al. 2016

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“…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)(7)(8) but can be grouped into 20 clusters and eight singletons, i.e., phages for which no close relatives have yet been identified (9,10). Genomic characterization of nearly 700 of these phages (http://phagesdb.org/) reveals a staggeringly large number of genes (Ͼ40,000) coding for products of unknown function.…”

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The Protein Interactome of Mycobacteriophage Giles Predicts Functions for Unknown Proteins

et al. 2015

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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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“…Mycobacteria, like other bacteria, may have the potential to exchange DNA. Indeed, experimental transduction has been performed in MTB [85], and natural conjugation has been demonstrated for M. smegmatis [143]. Nontuberculous mycobacteria can acquire antibiotic resistance genes from other species [80,140].…”

Section: Theoretical and Technical Assessmentmentioning

confidence: 99%

Pulmonary Tuberculosis and Mycobacterium Tuberculosis: Modern Molecular Epidemiology and Perspectives

Godreuil

Tazi

Bañuls³

2006

Encyclopedia of Infectious Diseases

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Molecular Genetics of Mycobacteriophages

Cited by 77 publications

References 217 publications

Fluorescent Reporter DS6A Mycobacteriophages Reveal Unique Variations in Infectibility and Phage Production in Mycobacteria

Fluorescent Reporter DS6A Mycobacteriophages Reveal Unique Variations in Infectibility and Phage Production in Mycobacteria

The Protein Interactome of Mycobacteriophage Giles Predicts Functions for Unknown Proteins

Pulmonary Tuberculosis and Mycobacterium Tuberculosis: Modern Molecular Epidemiology and Perspectives

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