Reversible bacteriophage resistance by shedding the bacterial cell wall

Introduction and background

All living organisms are attacked by viruses, and bacteria are no exception: they are infected by viruses called bacteriophages. To fight off viral invaders, bacteria have evolved various defence mechanisms. These include restriction-modification systems, abortive infection, and CRISPR-Cas systems. To invade the host, bacteriophages bind sugars and proteins present on the outside of the cell envelope. The cell envelope, composed of the cell wall and cell membrane, allows bacteria to withstand adverse environmental conditions, including changes in salt concentration, temperature, or pH, and serves as a selective barrier to the environment. Bacteriophages exploit molecules of the cell wall, using them as entry doors to penetrate the host cell.

In this study, Ongenae et al. describe a novel bacterial defence mechanism that blocks entry to phages: the temporary removal of their cell wall. This mechanism is tied to osmoprotective conditions – a situation in which the number of dissolved molecules in- and outside the cell stays constant. Such conditions exist in plant sap, in rainwater in soil or inside host cells – when bacteria reside inside mammalian cells.

The authors hypothesized that in these conditions, the lacking cell envelope provides no entry point for the bacteriophages, thereby preventing infection. These insights will help improve the use of therapeutics to fight bacterial infections. It is time to rethink the use of cell wall targeting therapeutics in osmoprotective conditions.

 

Findings

1. Distantly related bacteria defend themselves with a cell wall-deficient lifestyle

The authors tested their hypothesis in Streptomyces, a bacterium known for its cell wall-deficient lifestyle. Upon exposure to the lytic phage LA7 in osmoprotective conditions (LPB medium), Streptomyces formed cell wall-deficient (CWD) cells. The bacteria exhibited a survival rate of 4% compared with a control culture that died completely when exposed to the phage under non-osmoprotective conditions (LB medium). They determined the survival rate by counting the number of colony-forming units and observed that some bacterial cells regenerated a cell wall, indicating that cells could switch between both lifestyles. To confirm that their observations could generalize to other lytic phages, the researchers tested three other viruses (CE2, CE10, and LD10), which corroborated the previous result.

The researchers also wanted to know whether this defence strategy is shared with other bacterial species, and tested Bacillus subtilis and Escherichia coli, two species that are evolutionarily distant from Streptomyces, using φ29 and T4 bacteriophages respectively. They obtained the same results as described for Streptomyces. Previous research revealed that dying bacteria release endolysins – enzymes that degrade the cell wall. This could explain cell wall removal in surrounding bacteria. The scientists refuted this hypothesis: a phage-infected B. subtilis culture was filtered, and the supernatant including released endolysins from dying host cells was applied to a fresh bacterial culture. In the new culture, no CWD cells arose, confirming that endolysins from dying cells do not cause cell wall removal.

2.  The missing cell wall is key to the defence mechanism

To demonstrate that the absence of a cell wall and not another mechanism protects against viral attack, the scientists artificially generated CWD cells. They treated Streptomyces, B. subtilis, and E. coli with lysozyme and/or antibiotics to strip their cell wall. Subsequent phage infection had no effect on the bacteria, which shows that having no cell wall indeed protects against phage infection.

To confirm that phages fail to attach to CWD cells, the scientists used cryo-electron tomography (cryo-ET) and imaged the process. They obtained different results for E. coli and B. subtilis: no phages could attach to E. coli, while some bound the surface of B. subtilis. Two observations indicate that the φ29 phage successfully infected and proliferated in B. subtilis host cells: empty phage capsid heads, indicating that the bacteriophages successfully ejected their genetic material, and filaments that resembled p1 proteins, an indicator for ongoing phage DNA replication. These results suggest that artificial cell wall stripping protects E. coli against phage attachment but not B. subtilis.

To provide another line of evidence that phages are less successful in infecting and multiplying in CWD cells, the researchers assessed the phage’s progeny by counting the plaque forming units, equivalent to the number of new phages after proliferation in a bacterial culture. They counted less phage progeny in the LPB medium in B. subtilis and E. coli compared to control cultures in LB medium. This is consistent with the observation that bacteria have better chances of survival in LPB than in LB medium. Artificial cell wall stripping in E. coli effectively blocked all phage progeny compared to LPB medium, in which phages can still proliferate to a certain extend. This boost in protection through artificial stripping is not seen in B. subtilis. This suggest that artificial cell wall-stripping provides less protection to B. subtilis than to E. coli, which is in line with the cryo-ET data.

 

Importance and personal opinion

This preprint provides new insights into the toolbox that bacteria use to ward off viruses. It describes a novel mechanism that allows bacteria to defend themselves against various kinds of phages, in contrast to other mechanisms such as CRISPR-Cas systems, which only target specific phages.

This discovery is particularly valuable in the context of rising antibiotic resistances among pathogenic bacteria. One of the biggest medical challenges of the 21st century will be to develop new therapies to tackle bacterial infections. Phage therapy to treat bacterial infections is an avenue worth exploring. This study suggests that phage therapy and cell wall targeting antibiotics should not be used to tackle infections in osmoprotective environments.

Future research will have to focus on unravelling host-phage interactions, with the outlook to unveil new weak points for attack or to develop strategies to circumvent known defence mechanisms.

I find this preprint remarkable because it shows a novel bacterial defence mechanism. It also provides the first visualization of interaction between cell wall-free B. subtilis and bacteriophages using cryo-ET. It is delightful to see how cryo-ET enabled to capture the binding of bacteriophages to the bacterial cells and hijacking cellular machineries to replicate. This sets great expectations for what future studies will help visualize using this method.

 

Questions to the authors

• Do you have any ideas or guesses on how the cell wall shedding process begins? Could it be a “community answer”, as seen with quorum sensing?
• Do you have any plans on elucidating the signalling networks underlying cell wall removal? Could this be an interesting weak point to target with the goal to boost the effectivity of phage therapy?
• Do you think the difference in phage attachment in coli and B. subtilis CWD cells can be explained by the differences in their membrane structure?

 

 

Tags: antibiotic, bacterial defence, phage therapy

doi: https://doi.org/10.1242/prelights.31568

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