The project is proceeding very nicely, with substantial progress against all of the original main objectives.
Our first publication involved completing a line of research initiated during our previous ERC grant. Mercier et al., 2016 (Nature Microbiology 1, 16091) describe how we were able to isolate, for the first time, an E. coli mutant deleted for the normally essential key cell division gene, ftsZ, by starting with L-form cells. We characterised the mutations need to enable bypassing of ftsZ and showed that the basis for mutant survival was through a highly aberrant form of growth which we called “coliflower”. The results show how remarkably malleable bacterial cell morphology is and how simple in evolutionary terms the emergence of cells such as Mycoplasma, that can proliferate without FtsZ could have arisen.
Our first full research paper relating to work solely from the new grant, described the discovery of an unexpected gene in the cell wall synthesis pathway (Theme 1), and was also published in Nature Microbiology (Emami et al., 2016, Nature Microbiol. 2, 16253).
Our third paper described a major breakthrough in understanding how antibiotics and lytic enzymes, such as lysozyme, interact during L-form switching. We found that penicillin unexpectedly prevents the L-form switch in B. subtilis and probably a range of other organisms, including important pathogens, such as S. aureus. We then found that this effect can be overcome by treating cells with exogenous lysozyme. Amazingly, the lysozyme can be supplied by mammalian cells, such as macrophages, and again that this applies to both B. subtilis and pathogens. This work impinged on key objectives from all three themes of the original proposal. This paper was published in Cell (Kawai et al., Cell172, 1038–1104).
Our fourth paper solved the problem of why L-form growth often is best under anaerobic conditions. We found that blocking cell wall synthesis alters flux through the glycolytic pathway, which then leads to the oxidative stress that impairs growth (Kawai et al. 2019, Nature Microbiology).
The fifth paper (Mickiewicz et al. 2019, Nature communications 10, 379) directly addressed the important question of whether L-forms have a significant role to play in patients during recurrent infection. A detailed analysis of samples from a large cohort of patients suffering from recurrent urinary tract infections revealed the frequent presence of L-forms. We went on to characterise their properties including survival and ability to grow and proliferate in urine and to survive and divide in a zebra fish embryo model system.
In our 6th major paper (Wu et al., 2020, Nature Communications 11, 4149), we exploited the ability of L-forms to grow in microfluidic channels, which we had recently discovered, and reported a wide range of seminal observations about the effects of geometry on various cell cycle processes.
In addition to the research work we published several important review articles. First, a major review on the general topic of L-forms (Errington et al., 2016, Phil. Trans. Royal Soc. B 371, 20150494). A second major review focuses on bacterial membranes, incorporating a substantial section on L-forms (Strahl & Errington, 2017, Ann Revs Microbiol., 71, 519). The third review provides a personal perspective on the L-form story (Errington, 2017, Biochem. Soc. Transactions, 45, 287). Reviews Claessen and Errington (2019, Trends Microbiol. 27, 1025) and Westblade et al. (2020, PLOS Pathogens 16, e1008892) provide summaries and updates on the role of the L-form transition in adaptation to stress and in antibiotic tolerance, respectively. Errington and van der Aart (2020, Microbiology,166, 425) is a brief general review of B. subtilis including reference to the L-form transition. Finally, Egan et al. (2020, Nat Rev Microbiol. 18, 446) provides a comprehensive review of bacterial cell wall synthesis including the wall deficient L-form transition. It is going to be highly influential as it already has 88 citations (Google Scholar, 31 Aug 2021).
I have been commissioned to write a comprehensive review on bacterial L-forms for Nature Reviews Microbiology next year.
