The rising tide of antibiotic resistance among several important human pathogens is a major public health concern, and new complementary therapies that can help mitigate antibiotic resistance are urgently needed. In terms of their discovery, and therapeutic utilization, bacteriophages (viruses that infect bacteria) predate antibiotics by a few years. However, the effectiveness, and broad-spectrum activity of antibiotics triumphed over the narrow specificity of bacteriophages giving antibiotics a monopoly over antibacterial therapy early on in the 20^th century; this has continued to the present day. The current situation with difficult to treat infections involving bacteria that are resistant to multiple antibiotics has brought bacteriophages back into consideration as viable therapeutic agents. However, just as with antibiotics, bacteria can become phage resistant, and mutations in host factors required for any part of the phage life cycle can result in loss of infection.

The bacterial genus Mycobacterium encompasses infamous human pathogens such as Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, Mycobacterium leprae, the causative agent of leprosy, and several insidious opportunistic pathogens such as Mycobacterium abscessus and Mycobacterium avium. Ineffective treatments with antibiotics for M. abscessus have provided opportunities in the compassionate use of phages for therapy¹, and cocktails of bacteriophages could potentially be of use in treating extensively drug-resistant M. tuberculosis². To date the receptors and host factors required for phage infection of mycobacteria are largely unknown. Mutations in genes encoding proteins required by the phage but do not severely compromise bacterial fitness can result in phage resistance. Thus, if we are to use phages therapeutically, we need to learn more about how bacteria become resistant to phages and how the bacterial pathogen is affected by these changes.

For this work, we set out to find naturally occurring phage resistant mutants to discover host factors required for phage infection that may lead to phage resistance in Mycobacterium smegmatis, a harmless soil-dwelling cousin of the pathogenic Mycobacterium tuberculosis. We found that a few of the resistant mutants had mutations in the lsr2 gene that encodes the protein Lsr2. The Lsr2 nucleoid-associated protein is well conserved among the order actinobacteria but includes distant evolutionary relatives such as H-NS in E. coli. The Lsr2 protein is involved in global gene regulation and acts by binding specifically to A-T rich sites in the bacterial genome. A diverse array of phages that infect bacteria in the order Actinobacteria also encode their own copy of Lsr2-like proteins for reasons yet unknown. The lsr2 gene has been found to be essential for M. tuberculosis, where it is necessary for growth both in-vivo and in-vitro. Additionally, the Lsr2 protein is necessary for regulating expression of virulence factors in both pathogenic mycobacteria such as M. tuberculosis and M. leprae and the opportunistic pathogen M. abscessus.  

In this work, we employed state-of-the-art phage engineering utilizing CRISPR selection, with the previously described method CRISPY-BRED³ to create engineered lytic bacteriophages that either express fluorescent proteins during the course of the phage life cycle. Cutting-edge live-cell imaging techniques paired with microfluidics and cell and phage labeling probes allowed us to visualize the dynamics of the entire mycobacteriophage life cycle. We found that mycobacteriophages attach preferentially at sites of new cell wall deposition on the mycobacterial cell surface, where cells are actively growing. Additionally, we observe Lsr2 recruitment to spatially defined intracellular domains (zones of phage replication (ZOPR)), where mycobacteriophage assembly occurs. In bacteria where the lsr2 gene has been deleted, we see a marked defect in the formation of ZOPRs and an inability of phages to generate epidemics, causing resistance to various genetically distinct clusters of mycobacteriophages.

The novel tools and techniques we describe in the paper can be employed to find other host factors that result in phage resistance. These methods can be applied in the struggle against antibiotic-resistant M. tuberculosis and other troublesome mycobacteria.

1          Dedrick, R. M. et al. Phage Therapy of Mycobacterium Infections: Compassionate Use of Phages in 20 Patients With Drug-Resistant Mycobacterial Disease. Clinical infectious diseases 76, 103-112 (2023).

2          Guerrero-Bustamante, C. A., Dedrick, R. M., Garlena, R. A., Russell, D. A. & Hatfull, G. F. Toward a Phage Cocktail for Tuberculosis: Susceptibility and Tuberculocidal Action of Mycobacteriophages against Diverse Mycobacterium tuberculosis Strains. mBio 12, doi:10.1128/mBio.00973-21 (2021).

3          Wetzel, K. S. et al. CRISPY-BRED and CRISPY-BRIP: efficient bacteriophage engineering. Scientific Reports 11, 6796, doi:10.1038/s41598-021-86112-6 (2021).