Periodic Reporting for period 4 - ELFBAD (L-form bacteria, biotechnology and disease)
Periodo di rendicontazione: 2020-04-01 al 2021-06-30
The peptidoglycan cell wall is a defining structure of the bacteria. It is the target for our best antibiotics and fragments of the wall trigger powerful innate immune responses against infection. The genes for peptidoglycan synthesis are present in most bacterial lineages, suggesting that the wall emerged early in cellular evolution. Surprisingly, many bacteria can switch almost effortlessly into a cell wall deficient “L-form” state in which they become completely resistant to many cell wall active antibiotics. Remarkably, L-form growth is completely independent of the complex FtsZ-based division machine that is essential for proliferation of most bacteria. Proliferation occurs instead by a seemingly haphazard process involving membrane blebbing or tubulation and scission, leading to progeny of highly irregular size. The switch to this mode of proliferation seems to require only the upregulation of membrane synthesis, leading to an increased surface area to volume ratio. L-forms may provide insights into how primitive cells proliferated before the evolution of the cell wall and the ancient bacterial radiation. L-forms have also been implicated in many recurrent or chronic diseases, although these claims have been controversial. The key aims of our project were first, to improve our understanding of key features of the L-forms of our best characterised model system, B. subtilis, including both basic science and possible biotechnological applications. Second to extend our analysis of basic L-from biology into several diverse bacterial systems, of relevance to both biotechnology and infectious disease. Finally, to explore in detail the possible clinical relevance of L-forms, aiming to identify specific clinical situations in which they are relevant or, at least, to establish model systems in which the interactions between L-form and mammalian systems can be studied.
We made substantial progress against all of the objectives described above. Of particular importance, we showed how immune cells can drive the formation of L-forms, which are completely resistant to beta-lactam antibiotics and therefore could be responsible for infections being persistent or recurrent. We went on to show that we could detect abundant L-forms in patients with recurrent urinary tract infections, providing an important example in which L-forms may be supporting a clinically important role.
We made substantial progress against all of the objectives described above. Of particular importance, we showed how immune cells can drive the formation of L-forms, which are completely resistant to beta-lactam antibiotics and therefore could be responsible for infections being persistent or recurrent. We went on to show that we could detect abundant L-forms in patients with recurrent urinary tract infections, providing an important example in which L-forms may be supporting a clinically important role.
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.
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.
Our discoveries on the interaction of lysozyme and beta-lactam antibiotics on L-form biogenesis, and on the identification of L-forms in urinary tract infection patients could have a major impact on infectious disease research. We believe that many clinicians are now looking for L-forms in various chronic or recurrent infections, and may be able to find them using the methods we have developed.
We hope to be able to obtain proof of concept funding to develop L-forms for biotechnological applications. This again could have a very broad impact.
We hope to be able to obtain proof of concept funding to develop L-forms for biotechnological applications. This again could have a very broad impact.