Genome-wide CRISPRi mapping of mycobacteriophage interaction determinants in Mycobacterium smegmatis mc2155
- 1Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA; Biological Systems and Engineering, Lawre
Abstract
Bacteriophages are highly specific predators of bacterial hosts, but the molecular mechanisms governing their entry across the structurally 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, we mapped the functional architecture of host susceptibility determinants. This parallelized genetic screen revealed a spatial continuum of infection requirements spanning from outer leaflet surface structures down to deep cytoplasmic machinery. 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 identified 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 mycomembrane, arabinogalactan functions as a candidate secondary receptor 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. While physical deletions of major porins trigger spontaneous regulatory pathways that upregulate silent backup paralogues, acute transcriptional knockdowns circumvent suppressor accumulation, revealing unmasked single-gene dependencies. Structural analysis demonstrated that host determinant usage decouples entirely from whole-genome viral phylogeny, indicating that standard phylogenetic groupings fail to predict surface recognition profiles. Ultimately, mapping this host interactome reveals an evolutionary strategy where phages target highly redundant or abundant host machinery to exploit fixed physiological liabilities, presenting a robust molecular blueprint for the rational engineering of escape-resistant therapeutic phage cocktails.
Related
- Identification of novel inhibitors of Mycobacterium smegmatis growth through genome-wide overexpression of Cluster P3 mycobacteriophage Xavia genes
- Loss of function of LprG-Rv1410c and MmpL11 homologues in Mycobacterium smegmatis leads to altered glycopeptidolipid profile and decreased cellular c-di-GMP levels during biofilm formation
- Enhancing Mycobacterial Clearance via Heterologous BCG Prime Boost Strategy Using Recombinant Mycobacterium smegmatis
- Engineered Mycobacterium smegmatis-based intrapleural immunotherapy remodels the pleural immune microenvironment for malignant pleural effusion treatment
- High-throughput genetic interaction analysis in Mycobacterium tuberculosis identifies highly synergistic drug targets
BibTeX
@misc{maucourt2026genomewide,
title = {{Genome-wide CRISPRi mapping of mycobacteriophage interaction determinants in Mycobacterium smegmatis mc2155}},
author = {F. Maucourt and C. Lewis and A. Kazakov and I. Murray and D. Jacobs-Sera and A. M. Deutschbauer and K. S. Wetzel and M. Niederweis and G. F. Hatfull and V. K. Mutalik},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.09.757826},
url = {https://www.biorxiv.org/content/10.64898/2026.10.09.757826v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Genome-wide CRISPRi mapping of mycobacteriophage interaction determinants in Mycobacterium smegmatis mc2155
AU - F. Maucourt
AU - C. Lewis
AU - A. Kazakov
AU - I. Murray
AU - D. Jacobs-Sera
AU - A. M. Deutschbauer
AU - K. S. Wetzel
AU - M. Niederweis
AU - G. F. Hatfull
AU - V. K. Mutalik
PY - 2026
DA - 2026/10/09
PB - bioRxiv
DO - 10.64898/2026.10.09.757826
UR - https://www.biorxiv.org/content/10.64898/2026.10.09.757826v1
AB - Bacteriophages are highly specific predators of bacterial hosts, but the molecular mechanisms governing their entry across the structurally 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, we mapped the functional architecture of host susceptibility determinants. This parallelized genetic screen revealed a spatial continuum of infection requirements spanning from outer leaflet surface structures down to deep cytoplasmic machinery. 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 identified 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 mycomembrane, arabinogalactan functions as a candidate secondary receptor 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. While physical deletions of major porins trigger spontaneous regulatory pathways that upregulate silent backup paralogues, acute transcriptional knockdowns circumvent suppressor accumulation, revealing unmasked single-gene dependencies. Structural analysis demonstrated that host determinant usage decouples entirely from whole-genome viral phylogeny, indicating that standard phylogenetic groupings fail to predict surface recognition profiles. Ultimately, mapping this host interactome reveals an evolutionary strategy where phages target highly redundant or abundant host machinery to exploit fixed physiological liabilities, presenting a robust molecular blueprint for the rational engineering of escape-resistant therapeutic phage cocktails.
ER -