Phage resistance profiling identifies new genes required for biogenesis and modification of the corynebacterial cell envelope

Abstract
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Abstract

Bacteria of the order Corynebacteriales including pathogens such as Mycobacterium tuberculosi and Corynebacterium diphtheriae are characterized by their complex, multi-layered envelope. In addition to a peptidoglycan layer, these organisms possess an additional polysaccharide layer made of arabinogalactan and an outer membrane layer composed predominantly of long-chain fatty acids called mycolic acids. This so-called mycolata envelope structure is both a potent barrier against antibiotic entry into cells and a target of several antibacterial therapeutics. A better understanding of the mechanisms underlying mycolata envelope assembly therefore promises to reveal new ways of disrupting this unique structure for the development of antibiotics and antibiotic potentiators. Because they engage with receptors on the cell surface during infection, bacteriophages have long been used as tools to uncover important aspects of host envelope assembly. However, surprisingly little is known about the interactions between Corynebacteriales phages and their hosts. We therefore made use of the phages Cog and CL31 that infect Corynebacterium glutamicum (Cglu), a model member of the Corynebacteriales, to discover host factors important for phage infection. A high-density transposon library of Cglu was challenged with these phages followed by transposon sequencing to identify resistance loci. The analysis identified an important role for mycomembrane proteins in phage infection as well as components of the arabinogalactan and mycolic acid synthesis pathways. Importantly, the approach also implicated a new gene (cgp_0396) in the process of arabinogalactan modification and identified a conserved new factor (AhfA, Cpg_0475) required for mycolic acid synthesis in Cglu.

Data availability

Sequencing data generated from this study have been deposited in the NCBI Sequence Read Archive under BioProject PRJNA834153. All other data generated or analyzed during this study are provided in the manuscript and supporting files.

The following data sets were generated

1. McKitterick AC
2. Bernhardt TG
(2022) Phage resistance profiling identifies new genes required for biogenesis and modification of the corynebacterial cell envelope
NCBI BioProject, PRJNA834153.

https://www.ncbi.nlm.nih.gov/sra/PRJNA834153

The following previously published data sets were used

(2020) Phenotypic profiling of a Corynebacterium glutamicum transposon library
NCBI BioProject PRJNA610521.

https://www.ncbi.nlm.nih.gov/bioproject/610521

Article and author information

Author details

Amelia C McKitterick

Department of Microbiology, Howard Hughes Medical Institute, Harvard University, Boston, United States

Competing interests
The authors declare that no competing interests exist.
Thomas G Bernhardt

Department of Microbiology, Howard Hughes Medical Institute, Harvard University, Boston, United States

For correspondence
thomas_bernhardt@hms.harvard.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-3566-7756

Funding

Howard Hughes Medical Institute

Thomas G Bernhardt

Life Sciences Research Foundation

Amelia C McKitterick

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.