The phage-host interaction landscape of Mycobacterium smegmatis

Bacteriophages are highly specific predators of bacterial hosts, but the molecular mechanisms governing their entry across the complex mycobacterial cell envelope remain poorly defined. Utilizing a genome-wide CRISPR interference library in the model organism Mycobacterium smegmatis against a phylogenetically representative collection of 70 mycobacteriophages, and subsequent efficiency of plating assays, we mapped the host susceptibility determinants for phage infection. This screen established a spatial continuum of infection requirements spanning from the surface accessible outer membrane components down to cytoplasmic proteins. The four major outer membrane Mycobacterium smegmatis porins (Msp) emerged as the dominant candidate receptor family for 46 diverse phages, representing the first protein receptors proposed within this group. Interrogating the asymmetric outer bilayer also implicated cell envelope glycolipids as candidate receptors for 14 phages, resolving into glycopeptidolipid sub-lineages. Beneath the M. smegmatis mycomembrane, arabinogalactan functions as a determinant for 5 phages, while the inner membrane lipid transporter MmpL11 and the global nucleoid-associated protein Lsr2 represent determinant factors for 14 and 31 phages respectively. Although physical deletions of major porins genes trigger spontaneous regulatory pathways that upregulate silent backup paralogues, transcriptional knockdowns circumvent suppressor accumulation, revealing single-gene dependencies. Cluster analysis demonstrated that host determinant usage decouples from whole-genome viral phylogeny, indicating that standard phylogenetic groupings fail to predict surface recognition profiles. Ultimately, mapping these host dependencies reveals an evolutionary strategy where phages target highly redundant or abundant host proteins to exploit physiological liabilities, presenting a robust molecular blueprint for the rational engineering of escape-resistant therapeutic phage cocktails.