Defects in glycopeptidolipid biosynthesis confer phage I3 resistance in Mycobacterium smegmatis

Abstract

Mycobacteriophages have played an important role in the development of genetic tools and diagnostics for pathogenic mycobacteria, including Mycobacterium tuberculosis. However, despite the isolation of numerous phages that infect mycobacteria, the mechanisms of mycobacteriophage infection remain poorly understood, and knowledge about phage receptors is minimal. In an effort to identify the receptor for phage I3, we screened a library of Mycobacterium smegmatis transposon mutants for phage-resistant strains. All four phage I3-resistant mutants isolated were found to have transposon insertions in genes located in a cluster involved in the biosynthesis of the cell-wall-associated glycopeptidolipid (GPL), and consequently the mutants did not synthesize GPLs. The loss of GPLs correlated specifically with phage I3 resistance, as all mutants retained sensitivity to two other mycobacteriophages: D29 and Bxz1. In order to define the minimal receptor for phage I3, we then tested the phage sensitivity of previously described GPL-deficient mutants of M. smegmatis that accumulate biosynthesis intermediates of GPLs. The results indicated that, while the removal of most sugar residues from the fatty acyl tetrapeptide (FATP) core of GPL did not affect sensitivity to phage I3, a single methylated rhamnose, transferred by the rhamnosyltransferase Gtf2 to the FATP core, was critical for phage binding. INTRODUCTIONPhages that infect mycobacteria have played an important role in the development of genetic tools for mycobacteria, particularly Mycobacterium tuberculosis, which is the causative agent of tuberculosis Jacobs et al., 1991;Lee et al., 2004;Raj & Ramakrishnan, 1970;Snapper et al., 1988;van Kessel et al., 2008). Recombinant mycobacteriophages have also been used for phage typing of clinical isolates (Kubica, 1982;Rado et al., 1975), in diagnostics Hazbon et al., 2003; Jacobs et al., 1993;McNerney & Traore, 2005;Piuri et al., 2009;Riska et al., 1999), and as therapeutic agents for mycobacterial diseases (Broxmeyer et al., 2002;Mankiewicz & Beland, 1964). For the further development of mycobacteriophages as genetic tools for manipulation of mycobacteria, and as diagnostic and therapeutic agents, it is necessary to understand the mechanism of interactions between mycobacteriophages and mycobacteria. Attachment of a phage by binding to its receptor on the mycobacterial cell surface is important for initiating infection, and the study of phage-resistant mutants defective in phage adsorption is a useful approach to identifying these receptors. While a number of cell wall components have been implicated in roles as phage receptors (Besra et al., 1994;Bisso et al., 1976;Dhariwal et al., 1986;Furuchi & Tokunaga, 1972), no defined mycobacteriophage-resistant mutant with a missing phage receptor has been characterized to date. The only report of a mycobacteriophage-resistant mutant describes the appearance of a new lipid species (rather than the disappearance of an existing component) in a Mycobacterium smegmatis transposon mutant tha...

Mycobacteriophages

“…However, the key property of phages in such a cocktail is that resistance to one phage does not confer resistance to the other phages. Unfortunately, very little is known about either the specific receptors used for mycobacteriophage infection, or the mechanisms of resistance (132), and this is an area that needs further investigation. Furthermore, relatively few of the phages isolated on M. smegmatis infect M. tuberculosis , and they generally group in a small number of cluster or subclusters (84).…”

Molecular Genetics of Mycobacteriophages

“…However, in general, little is known about mycobacterial receptors for phage recognition or the determinants of host specificity. For phage I3 (which is not completely sequenced but is a Cluster C–like phage) resistance is accompanied by loss of cell-wall-associated glycopeptidolipids (121), and an M. smegmatis peptidoglycolipid, mycoside C(sm), has been implicated in the binding of phage D4 (which is also genomically uncharacterized) (122). Lyxose-containing molecules have also been proposed as possible receptors for the unsequenced phage Phlei (123, 124).…”

Cloning, mutagenesis, and characterization of the microalga Parietochloris incisa acetohydroxyacid synthase, and its possible use as an endogenous selection marker

“…There are some relationship between GPL and mycobacteriophage. It was reported that nsGPL was the receptor of mycobacteriophage D4 and I3, and the methylated Rha in GPLs was the active sit, while the mutant absent of GPLs remained sensitive to D29 and Bxz1 [Dhariwal et al, 1986; Chen et al, 2009]. The GPL‐deficient mutant of M. smegmatis was sensitive to mycobacteriophage D29 and TM4, indicating GPL is the selective receptor for the particular mycobacteriophage.…”