The kiss of death: Limosilactobacillus reuteri PKS drives intraspecies competition

The gut microbial ecosystem is governed by complex microbe-microbe interactions, with most documented as interspecies relationships. In this issue of Cell Host & Microbe, Özçam et al. identified intraspecies competition among related strains of the gut symbiont Limosilactobacillus reuteri that are mediated via the antimicrobial activity of polyketide synthase (PKS). The gut microbial ecosystem is governed by complex microbe-microbe interactions, with most documented as interspecies relationships. In this issue of Cell Host & Microbe, Özçam et al. identified intraspecies competition among related strains of the gut symbiont Limosilactobacillus reuteri that are mediated via the antimicrobial activity of polyketide synthase (PKS). Ecosystems are dynamic networks of species governed by a balance of cooperative behaviors and fierce competition over resources. The gut microbiota are no exceptions. In this environment constrained by limited space, variable levels of pH and oxygen, and periodical nutrient supplies, micro-organisms have developed various strategies to occupy a territory or secure access over limited resources. Collectively, this results in multidimensional microbe-microbe interactions that are challenging to study in such a complex community. Yet, it is fundamental to understand these interplays because of the tremendous influence that the gut microbial community exerts on its host's health. Indeed, the host's homeostasis relies on the maintenance of a symbiotic relationship with their gut microbiota, and thus it is crucial to understand the mechanisms that support resilience of this ecosystem, being in a symbiotic or a dysbiotic state (van de Guchte et al., 2018van de Guchte M. Blottière H.M. Doré J. Humans as holobionts: implications for prevention and therapy.Microbiome. 2018; 6: 81Crossref PubMed Scopus (91) Google Scholar). In this issue of Cell Host & Microbe, Özçam et al., 2022Özçam M. Oh J.H. Tocmo R. Acharya D. Zhang S. Astmann T.J. Heggen M. Ruiz-Ramírez S. Li F. Cheng C.C. et al.A secondary metabolite drives intraspecies antagonism in a gut symbiont that is inhibited by cell wall acetylation.Cell Host Microbe. 2022; 30: 824-835https://doi.org/10.1016/j.chom.2022.03.033Abstract Full Text Full Text PDF Scopus (2) Google Scholar used the common gut microbial symbiont Limosilactobacillus reuteri as a model to study intraspecies competition between closely related strains. Like interspecies competition, intraspecies competition can take two forms: (1) indirect competition occurs when individuals exploit the same resource until it becomes limiting for the others; and (2) direct competition occurs when an individual is actively fighting another to become dominant, which is also known as interference competition (Gilad, 2008Gilad O. Competition and Competition Models.in: Jørgensen S.E. Fath B.D. Encyclopedia of ecology. 2. Elsevier, 2008: 707-712Crossref Scopus (18) Google Scholar). Here, the authors explored specifically the mechanisms underlying direct competition in this well-characterized Gram-positive lactic-acid-producing species showing high intraspecies diversity (Nelson et al., 2010Nelson K.E. Weinstock G.M. Highlander S.K. Worley K.C. Creasy H.H. Wortman J.R. Rusch D.B. Mitreva M. Sodergren E. Chinwalla A.T. et al.A catalog of reference genomes from the human microbiome.Science. 2010; 328: 994-999Crossref PubMed Scopus (524) Google Scholar). In a previously published work, the same team had demonstrated that L. reuteri prophages provide a competitive advantage over non-producing strains, although it comes at a cost, reducing fitness of the prophage carrying strains (Oh et al., 2019Oh J.H. Lin X.B. Zhang S. Tollenaar S.L. Özçam M. Dunphy C. Walter J. van Pijkeren J.P. Prophages in Lactobacillus reuteri Are Associated with Fitness Trade-Offs but Can Increase Competitiveness in the Gut Ecosystem.Appl. Environ. Microbiol. 2019; 86 (e01922-19)Crossref PubMed Scopus (25) Google Scholar). Here, they focused their attention on gene clusters involved in the production of secondary metabolites because many of them act as antimicrobials. First, they demonstrated that L. reuteri strain R2lc outcompetes L. reuteri strain 100-23 in co-cultures due to a potent antimicrobial activity carried by the biosynthetic gene cluster polyketide synthase (PKS). These genes lead to the production of polyene compounds, which have been shown to drive antifungal activity. The authors thus hypothesized that pks produces a polyene-like compound that would exert direct antimicrobial effect on sensitive strains. This was confirmed following the observation that R2lc, but not R2lcΔpks mutant, produced a polyene-like compound that was not secreted in the extracellular medium, suggesting that cell-cell interaction is required for killing effect. The team then explored the behavior of these two strains in vivo by colonizing germ-free mice with either a 1:1 mixture of wild R2lc and 100-23 strains or with R2lcΔpks and wild type 100-23. Samples were collected along the entire gastrointestinal tract, from the forestomach—which is the natural ecological niche of L. reuteri in mice—to the colon and feces. In all collection sites, the Pks-expressing R2lc strain outcompeted the Pks-sensitive 100-23 strain, while this was not observed with R2lcΔpks, confirming that pks confers a competitive advantage in vivo. The authors further extended the exploration of the competitive advantage of strain R2lc over a set of 51 strains isolated from various hosts (i.e. rat, mouse, chicken, human, and pig). By running pairwise co-cultures with either the wild-type R2lc or R2lcΔpks, they observed that nearly 20% of the tested strains were naturally resistant to R2lc. Running a comparative genomic analysis of the resistant strains, they identified that they all carried a similar act gene encoding an O-acyltransferase. Next, they mined 133 genomes of L. reuteri isolates to identify related act genes. They determined that approximately 33% of these genomes contained an act gene predicted to encode an almost identical protein (over 99% amino acid identity), which they grouped in Clade I. To the contrary, Clade II and Clade III encoded an act-like gene with lower amino acid identity (below 72%) and were predicted to be Pks-sensitive. Therefore, this suggests that approximately 30% of L. reuteri isolates are naturally protected against pks expression. The authors further validated that act confers resistance to Pks in vitro and in vivo. For this purpose, they selected two act-containing strains from Clade I, the rat isolate mlc3, and the human isolate ATCC 6475 to run competition assays against Pks-producing R2lc. Interestingly, they observed that the mutant 6475Δact grew slower in vitro than the parent wild-type strain, while it was the opposite for the mlc3Δact mutant that grew faster than mlc3, indicating that act expression may be associated with a metabolic cost in a strain-specific manner. As expected, inactivation of act in these strains led to increased sensitivity to Pks-producing R2lc in vitro. In vivo, the competition was investigated only between R2lc±pks and mlc3±act. Consistent with in vitro results, act conferred resistance to Pks-mediated killing. Finally, the authors suspected that the act gene might exert its protective effect through acetylation of the peptidoglycan, since this is a common antimicrobial resistance mechanism already documented against ampicillin (Teethaisong et al., 2014Teethaisong Y. Autarkool N. Sirichaiwetchakoon K. Krubphachaya P. Kupittayanant S. Eumkeb G. Synergistic activity and mechanism of action of Stephania suberosa Forman extract and ampicillin combination against ampicillin-resistant Staphylococcus aureus.J. Biomed. Sci. 2014; 21: 1-11Crossref Scopus (34) Google Scholar). Using the Pks-sensitive 100-23 strain that does not encode Act and cloning the act gene into it, the authors demonstrated that act expression led to a significant increase in total acetylation levels of the cell wall. Consistently, inhibition of act in Pks-resistant strains resulted in a drastic decrease in total acetylation levels. Together, this indicates that Act promotes acetylation of L. reuteri's cell wall. Yet, the exact mechanisms of interaction between Pks and Act remain to be fully elucidated. Hence, the authors provide here evidence of a strain-specific mechanism of intraspecies competition at play in the mammalian gut driven by pks and act genes. Pks is located on a plasmid, a mobile element that can be discarded by bacteria when it is not necessary to save the metabolic cost of pks expression. In fact, the metabolic tradeoff of pks expression was illustrated by the faster growth rate of R2lcΔpks compared with the parent wild-type strain. To the contrary, act is located on the bacterial chromosome, suggesting that it has been selected by the evolutionary process to be incorporated into the bacterial genome. In addition, as pointed out by the authors, act is widely distributed among L. reuteri strains isolated from evolutionarily distant hosts, such as humans and rodents, which suggests that evolution has favored act because it likely improves bacterial fitness in the gut. Interestingly, the act gene also provides some protection against the host's lysozyme enzyme, an abundant antimicrobial protein secreted by all mammals that kills bacteria by hydrolyzing the peptidoglycan and disturbing the stability of the cell membrane (Ragland and Criss, 2017Ragland S.A. Criss A.K. From bacterial killing to immune modulation: Recent insights into the functions of lysozyme.PLoS Pathog. 2017; 13e1006512Crossref PubMed Scopus (388) Google Scholar). As a consequence, the host itself also exerts an additional selective pressure on the expression of act. Therefore, considering the intense selective pressure exerted on act, it is rather surprising that only 30% of L. reuteri strains conserved act in their genome. A possible explanation is that act is not always required to survive Pks-mediated competition in complex biofilms, as was illustrated by the study herein, where it was observed that some act negative strains were nevertheless Pks resistant. As a matter of fact, the work by Özçam et al., 2022Özçam M. Oh J.H. Tocmo R. Acharya D. Zhang S. Astmann T.J. Heggen M. Ruiz-Ramírez S. Li F. Cheng C.C. et al.A secondary metabolite drives intraspecies antagonism in a gut symbiont that is inhibited by cell wall acetylation.Cell Host Microbe. 2022; 30: 824-835https://doi.org/10.1016/j.chom.2022.03.033Abstract Full Text Full Text PDF Scopus (2) Google Scholar only explored act-mediated protection in simplified models using pair-wise strain comparison in vitro and in vivo in gnotobiotic mice. Therefore, it remains to investigate to what extent pks and act both provide a competitive advantage within a complex ecosystem. Overall, this inspiring study contributes to provide better understanding of microbe-microbe-host interactions, which are so challenging to study, although deeply needed to refine microbiome-targeted interventions aiming at restoring symbiosis with the host in the context of diseases. S.P.C. is an employee of Ysopia bioscience with managerial responsibilities. A secondary metabolite drives intraspecies antagonism in a gut symbiont that is inhibited by cell-wall acetylationÖzçam et al.Cell Host & MicrobeApril 19, 2022In BriefÖzçam et al. show that L. reuteri strains encode an antimicrobial polyketide (Pks), which provides a competitive advantage in the gut. Select strains that can co-exist with L. reuteri encode a conserved acyltransferase (Act), which acetylates cell-wall components. Wild type, but not Δpks, outcompetes act mutants in vivo. Full-Text PDF Open Access