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

Abstract

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...

Arrayed CRISPRi and quantitative imaging describe the morphotypic landscape of essential mycobacterial genes

“…The biological function of this system is currently unclear; however, one practical implication is that, if involved as expected in intrinsic phage defense, it might inadvertently eliminate phages, which could be otherwise useful for clinically relevant mycobacteria such as M. tuberculosis or M. abscessus ( Dedrick et al, 2019 ). It is tempting to consider, too, if the differential complement of R-M systems among mycobacteria might determine the apparent species-selectivity of some mycobacteriophages – for example, DS6A ( Mayer et al, 2016 ) – and whether the MSMEG_3213 methylase, which is conserved in M. tuberculosis , functions in cell-cycle regulation ( Wion and Casadesús, 2006 ) in addition to its role in self-defense.…”

Engineered K1F bacteriophages kill intracellular Escherichia coli K1 in human epithelial cells

“…We inserted a gfp gene into the phage genome by combining in vivo homologous recombination with CRISPR/Cas-selection. The engineering of fluorescent bacteriophages with the insertions of a fluorescent gene in their genome has been applied before, using various methodologies and different bacteriophages, such as T7 51 , PP01 52 , T4 53 , P22 54 , 55 as well as the mycobacteriophages D29 56 and DS6A 57 , where the fluorescent protein is either expressed in the capsid of the phage or in a non-essential region of its genome. We report for the first time the engineering of a fluorescent phage K1F and we have applied for the first time CRISPR/Cas to select for fluorescent bacteriophages.…”

Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of Mycobacterium tuberculosis through Nutrient Auxotrophy

“…The ability of the BSL2-safe M. tuberculosis strains mc 2 7901 and mc 2 7902 to serve as mycobacteriophage hosts was therefore examined. The mycobacteriophages phAE912 ( 16 ), a DS6A phage restricted to the M. tuberculosis complex and expressing mVenus, and Φ 2 DRM9 ( 17 ), which infects both M. smegmatis and M. tuberculosis , infected mc 2 7901 and mc 2 7902 ( Fig. 3B and C ).…”