L-form conversion in Gram-positive bacteria enables escape from phage infection

Abstract

At the end of a lytic bacteriophage replication cycle in Gram-positive bacteria, peptidoglycan-degrading endolysins that cause explosive cell lysis of the host can also attack non-infected bystander cells. Here we show that in osmotically stabilized environments, Listeria monocytogenes can evade phage predation by transient conversion to a cell wall-deficient L-form state. This L-form escape is triggered by endolysins disintegrating the cell wall from without, leading to turgor-driven extrusion of wall-deficient, yet viable L-form cells. Remarkably, in the absence of phage predation, we show that L-forms can quickly revert to the walled state. These findings suggest that L-form conversion represents a population-level persistence mechanism to evade complete eradication by phage attack. Importantly, we also demonstrate phage-mediated L-form switching of the urinary tract pathogen Enterococcus faecalis in human urine, which underscores that this escape route may be widespread and has important implications for phage- and endolysin-based therapeutic interventions.

Competition between lysogenic and sensitive bacteria is determined by the fitness costs of the different emerging phage-resistance strategies

“…In agreement with this hypothesis, we found that capsule inactivation in ST14 is associated with an insertion of a thymine among a repeat of thymine residues ( Supplementary file 2 ). Other, non-genetic mechanisms of transient resistance to phages have also been described ( Bull et al, 2014 ; Cota et al, 2015 ; Ongenae et al, 2022 ; Wohlfarth et al, 2023 ). One is the epigenetic-based resistance based on DNA modifications, such as methylation.…”

“…This was previously shown to regulate the length of the O-antigen length by phase variation in Salmonella enterica , and resulted in transient phage resistance ( Cota et al, 2015 ). Additionally, transient cell wall shedding in filamentous actinobacteria, B. subtilis and E. coli ( Ongenae et al, 2022 ) and conversion to cell wall-deficient L-forms in Listeria monocytogenes ( Wohlfarth et al, 2023 ) can lead to transient phage resistance. An analogous process in K. pneumoniae could involve the shedding of the capsule in response to phage pressure, resulting in a state of reduced phage adsorption and limited infection.…”

The reproduction of Gram-negative protoplasts and the influence of environmental conditions on this process

“…In contrast to our conventional understanding, some recent studies showed that the transformation of bacteria into their protoplast state could happen in natural environments without antibiotic stress 6,16 . This transformation is observed to have been induced by environmental conditions like osmolarity and exposure to lytic phases 6,16 .…”

“…In contrast to our conventional understanding, some recent studies showed that the transformation of bacteria into their protoplast state could happen in natural environments without antibiotic stress 6,16 . This transformation is observed to have been induced by environmental conditions like osmolarity and exposure to lytic phases 6,16 . This transformation of cells into the protoplast state is shown to confer cells with phase resistance, increased survival rates in natural environments, and receptivity to incorporating DNA from the surrounding environment 6,7,17 .…”

“…However, bacteria are known to lose their cell wall and transform into their protoplast state when exposed to cell wall-degrading enzymes and bactericidal antibiotics 3,4 . Although this transformation is thought to make cells susceptible to changes in environmental conditions like osmolarity 5 , it is also known to confer some advantages to the bacteria 3,6,7 . Most of the antibody-binding sites are located on the surface of the cell wall 8,9 .…”