Substrate stiffness impacts early biofilm formation by modulating Pseudomonas aeruginosa twitching motility
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Curated by eLife
eLife assessment
This study connects changes in single-cell twitching motility due to substrate stiffness to multicellular phenotypes. It is likely to have a broad impact on those studying microbiology and multicellular communities as it assesses the influence of single-cell behavior on multicellular processes. However, some of the presented data conflict with previously published literature, raising questions about the nature of these differences.
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Abstract
Surface-associated lifestyles dominate in the bacterial world. Large multicellular assemblies, called biofilms, are essential to the survival of bacteria in harsh environments and are closely linked to antibiotic resistance in pathogenic strains. Biofilms stem from the surface colonization of a wide variety of substrates encountered by bacteria, from living tissues to inert materials. Here, we demonstrate experimentally that the promiscuous opportunistic pathogen Pseudomonas aeruginosa explores substrates differently based on their rigidity, leading to striking variations in biofilm structure, exopolysaccharides (EPS) distribution, strain mixing during co-colonization and phenotypic expression. Using simple kinetic models, we show that these phenotypes arise through a mechanical interaction between the elasticity of the substrate and the type IV pilus (T4P) machinery, that mediates the surface-based motility called twitching. Together, our findings reveal a new role for substrate softness in the spatial organization of bacteria in complex microenvironments, with far-reaching consequences on efficient biofilm formation.
Article activity feed
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eLife assessment
This study connects changes in single-cell twitching motility due to substrate stiffness to multicellular phenotypes. It is likely to have a broad impact on those studying microbiology and multicellular communities as it assesses the influence of single-cell behavior on multicellular processes. However, some of the presented data conflict with previously published literature, raising questions about the nature of these differences.
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Reviewer #1 (Public Review):
The authors present a very nice and timely study detailing how single Pseudomonas aeruginosa cells develop into microcolonies. They demonstrate that motility differences from changes in substrate stiffness are likely responsible for differences in microcolony morphology exhibited at different stiffnesses. The authors further conclude based on modeling data that these motility changes are not due to physiological changes resulting from surface sensing, but rather that mechanical properties of the substrate are responsible for modulating motility differences. However, this conclusion is derived partly from the use of a chpA mutant, which the authors' data demonstrate does not exhibit differences in motility compared to WT. These data are very surprising given that several published studies demonstrate a defect …
Reviewer #1 (Public Review):
The authors present a very nice and timely study detailing how single Pseudomonas aeruginosa cells develop into microcolonies. They demonstrate that motility differences from changes in substrate stiffness are likely responsible for differences in microcolony morphology exhibited at different stiffnesses. The authors further conclude based on modeling data that these motility changes are not due to physiological changes resulting from surface sensing, but rather that mechanical properties of the substrate are responsible for modulating motility differences. However, this conclusion is derived partly from the use of a chpA mutant, which the authors' data demonstrate does not exhibit differences in motility compared to WT. These data are very surprising given that several published studies demonstrate a defect in both pilus synthesis and twitching motility in PilChp mutants (including chpA). It is unclear what the differences are between the presented study and the published literature leading to the disparity in these results.
Major strengths of the manuscript include the detailed analysis of differences in phenotypes on substrates with different rigidities and a link back to changes in motility at the single cell level that could describe these differences.
A weakness of the manuscript is the difference between reported motility phenotypes here and what has been previously published in the literature.
Should the above confounding results be clarified, this work will have a broad impact on the field of microbiology and those studying complex microbial communities as it connects relevant phenotypic differences at the single cell level to mechanical perturbations and multicellular morphologies.
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Reviewer #2 (Public Review):
In this manuscript, Gomez et al. study the role of substrate stiffness in the first steps of biofilm formation of the versatile pathogen Pseudomonas aeruginosa. In a very thorough experimental set-up, the authors demonstrate that the early colonization of surfaces by Pseudomonas aeruginosa depends on the surface stiffness, irrespective of the chemical nature of the surface. At low stiffness, the bacteria form dense microcolonies, move slowly, do not explore most of the surface, and excrete minimal amounts of extracellular matrix. On the other hand, at high stiffness, the bacteria cover most of the available surface more uniformly, move rapidly, and excrete large amounts of extracellular matrix polymers. The surface stiffness doesn't affect the division time, but the residence time of bacteria in the constant …
Reviewer #2 (Public Review):
In this manuscript, Gomez et al. study the role of substrate stiffness in the first steps of biofilm formation of the versatile pathogen Pseudomonas aeruginosa. In a very thorough experimental set-up, the authors demonstrate that the early colonization of surfaces by Pseudomonas aeruginosa depends on the surface stiffness, irrespective of the chemical nature of the surface. At low stiffness, the bacteria form dense microcolonies, move slowly, do not explore most of the surface, and excrete minimal amounts of extracellular matrix. On the other hand, at high stiffness, the bacteria cover most of the available surface more uniformly, move rapidly, and excrete large amounts of extracellular matrix polymers. The surface stiffness doesn't affect the division time, but the residence time of bacteria in the constant flow configuration used in the paper is longer on stiffer substrates. Ultimately, the substrate stiffness differences lead to differences in gene expression. The carefully executed experiments are interpreted in the light of interesting simple models that help illuminate the wealth of information presented. The overall subject of the role of rigidity in bacterial physiology is topical and should be of interest to many scientists. The fact that a model without any explicit mechanosensing via Type IV pili can still account for the substrate stiffness phenotypic differences in colonization is a superb addition to the field and is fully supported by the data presented. Yet, some additional explanations will help even strengthen the work.
One of the difficulties in navigating the paper as it stands is the definition of many parameters in a global manner as fits from derived equations whose assumptions are not always fully validated. For instance, Equation (1) assumes no new addition because of the flushing of the channel with the clean medium. Yet the first peak of residence time on 2.7 kPa gels is around 5 minutes per Fig. S7 whereas the calculation of Vg is done over 100 minutes which should leave plenty of time for detachment and reattachment of bacteria upstream of the recording field of view, no? Similarly, the definition of Vcm is not easy to follow or apprehend. Is it that the general averages of the velocities are too noisy?
While the simple kinetic model presented does encapsulate many of the aspects of the data in an understandable way, some of the assumptions should be discussed further. Nowhere is it more important than in the assumption that pili only binds with its tips. While this assumption allows many simplifications in the model, type IV pili can potentially bind throughout their length, and as they can be microns in length, so can the binding region. The Koch et al 2021b does go over the reasoning but having a small discussion earlier in the paper would be great.
One of the very interesting characteristics of the models put forth is that they do not rely on direct mechanosensing from the bacterial side but rather are an indirect consequence of substrate rigidity and pili dynamics. The authors mention that the Pil-Chp and Wsp systems are the only ones found so far in Pseudomonas, but this doesn't mean that there is not another system in place. Making clear that they do not fully rule out the possibility of mechanosensing would be interesting.
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