Interdisciplinary life of microbes: from single cells to multicellular aggregates

Bacterial swarming is a rapid mass-migration, in which thousands of cells spread collectively to colonize surfaces. Physically, swarming is a natural example for active particles that use energy to generate motion. Accordingly, understanding the constraints physics imposes on these dynamics is essential for understanding the mechanisms underlying swarming. We present new experiments of swarming Bacillus subtilis mutants with different aspect ratios and at different densities; two physical quantities known to be associated with collective behavior. Analyzing the dynamics reveals a rich phase diagram of qualitatively distinct swarming regimes, describing how cell shape and population density govern the dynamical characteristics of the swarm. In particular, we show that under standard conditions, bacteria inhabit a region of phase space that is associated with rapid mixing and robust dynamics, with homogeneous density and no preferred direction of motion. The results suggest that bacteria have adapted their physical properties to optimize the principle functions assumed for swarming.

An overarching theme of cellular regulation in bacteria arises from the trade-off between growth and stress resilience. In addition, the formation of biofilms contributes to stress survival, since these dense multicellular aggregates, in which cells are embedded in an extracellular matrix of self-produced polymers, represent a self-organized protective and homeostatic 'niche'. As shown here for the model bacterium E. coli, the inverse coordination of bacterial growth with survival and the transition to multicellularity is achieved by a highly integrated regulatory network with several sigma subunits of RNA polymerase and a small number of transcriptional hubs as central players. By conveying information about the actual (micro)environments, nucleotide second messengers such as cAMP, (p)ppGpp and in particular c-di-GMP are the key triggers and drivers that promote either growth or stress resilience as well as organized multicellularity in a world of limited resources. Hengge, R. (2020) Linking bacterial growth, survival and multicellularity - small signaling molecules as triggers and drivers. Curr. Opin. Microbiol. 55: 57-66.

Life on earth is heavily based on chemical communication between cells. Quorum sensing enables unicellular organisms to coordinate their behavior and function in such a way that they can adapt to changing environments and compete, as well as coexist, with multicellular organisms. Prime examples of this phenomenon are displayed by the opportunistic pathogens Pseudomonas aeruginosa and Agrobacterium tumefaciens, which cause disease in humans and plants, respectively – but most often don’t. Quorum sensing in these pathogens is mediated by small amphiphilic signaling molecules such as 3-oxo-C12-HSL and 3-oxo-C8-HSL, leading to biofilm formation and secretion of virulence factors. The Meijler group is targeting QS in various pathogens with several chemical tools, such as a set of electrophilic and photoactivatable ‘tag-free’ probes that are designed to bind QS receptors covalently. These probes are used as molecular tools to obtain new insights into the mechanisms of activation, deactivation and recognition of bacterial quorum sensing – thtough the use of bioorthogonal chemistry. Diverse eukaryotes have been found to react strongly to the presence of these compounds, and the recognition of QSMs is mediated by mostly unknown receptors. Recently we identified and validated the role of a human receptor for HSLs, called the Major Vault Protein (MVP), as an important immunomodulator.

In nature, bacteria primarily live in surface-attached communities termed biofilms, in which cells are attached to each other through an extracellular matrix. These biofilms represent the most abundant form of biomass on our planet. In this presentation, I will first introduce microscopy and image processing techniques that enable us to monitor all individual cells in living biofilms. Based on these techniques, I will then show how we can identify the cell-cell interaction processes that determine the architecture development of biofilm microcolonies, across different species. I will then proceed to discuss how we use high-dimensional microstsate measurements to identify cell-cell interactions that dominate during the development from biofilm microcolonies to macrocolonies, with an emphasis on metabolic interactions. The techniques presented in this talk are broadly applicable for the analysis of spatially structured bacterial biofilm communities, and the conclusions I will present for dominant cell-cell interactions during biofilm development are likely to be conserved across species.

At the level of single bacteria, membrane potential is surprisingly dynamic and transient hyperpolarization has been associated with increased death rate. Yet, little is known about the spatiotemporal dynamics of membrane polarization during biofilm development. Here, we reveal a discrete transition from uncorrelated to collective polarization dynamics within spherical colonies. Suddenly, a shell of hyperpolarized cells forms at the colony centre and hyperpolarization travels radially outward. In its wake, bacteria depolarize, reduce their growth rate, and become tolerant against antibiotics, indicating the onset of habitat diversity. Single cell live imaging and modelling link hyperpolarization to an oxygen gradient formed within the colonies. We anticipate that dynamical polarization patterns are tightly connected to biofilm differentiation in various bacterial species.

The molecular basis of the run-and-tumble motility of {\it Escherichia coli} has been studied extensively. However, the quantitative spatiotemporal characterization of a swimming population remains challenging because of the broad range of relevant length and time scales. We show how this challenge can be met by using renewal processes to analyze measurements of the intermediate scattering function over length scales from $\sim10^0-10^2\mu$m. This allows us to demonstrate quantitatively how the persistence length of an engineered strain can be controlled by a chemical inducer and to characterize a transition from perpetual tumbling to smooth swimming. For wild-type {\it E.~coli}, we quantitatively bridge for the first time small-scale directed swimming and large-scale diffusion, hence measuring simultaneously motility parameters and the effective diffusivity.

Microbes as single-cell organisms are important model systems to study cellular mechanisms and functions. In recent years and with the help of advanced fluorescence microscopy techniques, immense progress has been made in characterizing and quantifying the behavior of single bacterial cells on the basis of molecular interactions and assemblies in the complex environment of live cultures. Importantly, single-molecule imaging enables the in vivo determination of the stoichiometry and molecular architecture of subcellular structures, yielding detailed, quantitative, spatiotemporally resolved molecular maps and unraveling dynamic heterogeneities and subpopulations on the subcellular level. Nevertheless, open challenges remain. Here, I quickly review the past and current status of the field, discuss example applications from our own work and give insights in future trends [1, 2]. References: [1] Vojnovic, I., Winkelmeier, J. and Endesfelder, U., Biochemical Society Transactions 2019, 10.1042/BST20180399 [2] Endesfelder, U., Essays in Biochemistry 2019, 10.1042/EBC20190002

Natural isolates of the endospore-forming model bacterium Bacillus subtilis show complex multicellular traits. Similar to plants, out of a spore of B. subtilis a 3D macroscopic multicellular structure can emerge that produces many more spores - a fruiting body. Different cell types partake in the formation of a fruiting body and its substructures. We will outline our approach to decipher how cell-type specific functions support biofilm formation using a combination of different imaging technqiues and computer-based image analysis methods. With this approach we want to test different hypotheses regarding the formation of fruiting bodies by employing targeted genetic manipulations of B. subtilis to reveal whether and how these genetic elements contribute to emergent properties during multicellular development.

Bacterial cell-cell signaling allows for coordinated cellular response through the secretion and detection of small diffusible signal molecules. While many quorum-sensing bacteria grow in spatially structured and genetically heterogeneous communities, the principles that govern quorum-sensing signaling under these conditions are largely unknown. In this lecture, I present recent results from our lab, where we have characterized the range of signaling between bacteria and explored its implication for regulation of horizontal gene transfer and for the formation of synthetically engineered spatial patterns. By combining microfluidic experiments with mathematical modeling, we quantify signal propagation in synthetic bacterial communities at a single-cell resolution. We identify two generic quorum-sensing designs that profoundly differ in the spatial scale of their effect: one design allows for global communication among cell clusters in the community, whereas the other design allows localized communication with signaling length-scale of about 10 microns. We further show that several mobile genetic elements, including phages and conjugative elements, employ self-limiting quorum sensing design to adaptively trigger horizontal gene transfer. This allows them to determine the fraction of uninfected host cells in a highly localized neighborhood and initiate horizontal gene transfer when this fraction increases. Finally, we constructed a synthetic gene network based on short range signaling which leads to pattern formation at the cellular scale.

Motile and sessile bacterial lifestyles are shaped by a combination of physical and biological processes. One example where this combination can be studied is magnetotaxis or magneto-aerotaxis, where magnetotactic bacteria navigate along field lines of a magnetic field, often the magnetic field of the Earth. In complex environments such as sediments, magnetotactic motility is not only determined by the magnetic field, but also by interactions with/responses to surfaces and active directional changes. I will show experimental and computational results illustrating this interplay for two systems, confinment to small spaces and sediment-like environments. Time permitting, I may also touch upon other topics of our group, specifically the motilie/biofilm switch in Bacillus subtilis and the collective behavior of cyanobacterial filaments.

The immunological control of infections with bacterial or protozoan pathogens that reside in macrophages depends on the differentiation and expansion of T lymphocytes that are capable to trigger antimicrobial mechanisms within the host cells. In the past, the analysis of the underlying pathways mainly focused on the critical immune cell components and products as well as on the signalling cascades that are necessary to elicit the macrophage effector functions. In this presentation, I will highlight recent insights into the microenvironmental and metabolic factors that strongly affect the immune response. Using infections with the vector-borne, flagellated protozoan parasite Leishmania as an example, I will demonstrate how the availability of a single amino acid influences the quality of the immune response and thereby accounts for the outcome of a cutaneous infection and how this can be utilized for novel therapeutic interventions.

The three most salient features of COVID-19 are its lethality for a significant human subpopulation, its high infectivity, and its heterogeneity of outcomes, ranging from essentially no symptoms to death. Its lethal pathologies include 1) amplified forms of inflammation (Acute Respiratory Distress Syndrome (ARDS), cytokine storms, septic shock) and 2) dysregulated forms of coagulation (severe blood clots that lead to cardiac events, multi system inflammatory syndrome in children (MIS-C)). At present, it is not clear how these consequences are induced, or how they relate to in milder pathologies observed in the clinic, such ‘COVID fingers’ or arthritis-like syndromes. By combining machine learning, synchrotron structural studies, computer simulations, in vitro cell based experiments, in vivo mouse experiments, and analysis of human COVID patient samples, we show how proteolytic processing of SARS-CoV-2 can potentially precipitate these outcomes, via a novel form of biomimicry that results in grossly distorted immune responses, coagulation pathologies, and suppression of type I interferon-based antiviral defenses, whereas that of other non-pandemic coronaviruses cannot.

Gliding filamentous cyanobacteria provide an experimental realization of long, flexible, and self-propelled polymers on surfaces. In addition, their motion is influenced by direction reversal and responses to light. To study the patterns formed by these active polymers, we combine simulations and experiments of cyanobacteria in quasi-2D confinement. At high densities of the filaments, our simulation shows dense swarms of filaments with both polar and nematic order. The long-range nematic and polar order are also confirmed by the experiments.

One of the debilitating causes of high mortality in the case of tuberculosis and other bacterial infections is the resistance development against standard drugs. There are limited studies so far to describe how bacterial second messenger signaling can directly participate in precise antibiotic tolerance characteristics of a cell in a target-dependent manner. In this study, we probe the role of secondary messenger c-di-AMP in drug tolerance, which includes both persister and resistant mutant characterization of Mycobacterium smegmatis. Specifically, with the use of c-di-AMP null and overproducing mutants, we showed how c-di-AMP plays a significant role in resistance mutagenesis against antibiotics with different mechanisms of action. We elucidated the specific molecular mechanism linking the elevated intracellular c-di-AMP level and high mutant generation and highlighted the significance of non-homology-based DNA repair. Further investigation enabled us to identify the unique mutational landscape of target and non-target mutation categories linked to intracellular c-di-AMP levels. Overall fitness cost of unique target mutations was estimated in different strain backgrounds, and then we showed the critical role of c-di-AMP in driving epistatic interactions between resistance genes, resulting in the evolution of multi-drug tolerance. Finally, we identified the role of c-di-AMP in persister cells regrowth and mutant enrichment upon cessation of antibiotic treatment. Additionally, we have proposed multiple mechanisms for how the second messenger molecule c-di-AMP moderates antibiotic sensitivity and resistance profile of M. smegmatis against different drugs. Our data also suggested how intracellular c-di-AMP concentration influence some surface-related properties including cellular aggregation, sliding motility, and Biofilm/Pellicle formation in M. smegmatis. The implications of these findings will be helpful in the context of understanding the link between second messenger signaling with biofilm formation and antimicrobial resistance.

Amyloids are protein fibers with unique and strong structures, known mainly in the context of neurodegenerative diseases. Surprisingly, amyloid fibers are secreted by species across kingdoms of life, including by microorganisms, and helps their survival and activity. Our laboratory published the first molecular structures of functional bacterial amyloid fibrils, which serve as key “weapons” making infections more aggressive. Some of these fibrils stabilize extremely strong and resistant layers of bacteria called biofilms. Others are cytotoxic to human immune cells. Thereby, they exposed new routes for the development of novel antivirulence drugs, which may elicit less resistance as the evolutionary pressure on the microbe is less profound compared to bactericidal drugs. Moreover, we revealed that amyloids secreted by bacteria highly abundant in the microbiome and food sources show similarities in molecular structures to human amyloids involved in neurodegenerative diseases such as Alzheimer’s and Parkinson’s. This might raise concerns about the involvement of microbes in facilitating these diseases, similar to prion proteins transmitted by contaminated meat that elicit the Creutzfeldt-Jakob disease. In addition, we identified peptides produced across species that provide antimicrobial protection that form amyloid fibrils, and determined their first high resolution structures. This amyloid-antimicrobial link signifies a physiological role in neuroimmunity for human amyloids. Such antimicrobial fibrils can serve as a stable coating for medical devices or implants, industrial equipment, food packing and more. The findings also provided atomic-level validation to the connection between antibacterial activity and amyloids formation, supporting a physiological role for amyloids, otherwise associated with disease, in fighting microbial threat to the brain. Key References 1. E. Tayeb-Fligelman, O. Tabachnikov, A. Moshe, O. Goldshmidt-Tran, M.R. Sawaya, N. Coquelle, J-P. Colletier, and M. Landau. The Cytotoxic Staphylococcus aureus PSMα3 Reveals a Cross-α Amyloid-like Fibril. Science 355(6327): 831-833; 2017 2. N. Salinas, A. Moshe, J-P. Colletier, and M. Landau. Extreme Amyloid Polymorphism in Staphylococcus aureus Virulent PSMα Peptides. Nat. Commun. 9(1):3512; 2018. 3. S. Perov, O. Lidor, N. Salinas, N. Golan, E. Tayeb-Fligelman, M. Deshmukh, D. Willbold, and M. Landau. Structural Insights into Curli CsgA Cross-β Fibril Architecture Inspired Repurposing of Anti-amyloid Compounds as Anti-biofilm Agents. PLoS Pathog 15(8): e1007978; 2019 4. Y. Engelberg, and M. Landau. The Human LL-37(17-29) Antimicrobial Peptide Reveals a Functional Supramolecular Nanostructure. Nat. Commun. 11, 3894; 2020 5. N. Salinas, E. Tayeb-Fligelman, M. Sammito, D. Bloch, R. Jelinek, D. Noy, I. Uson, and M. Landau. The Amphibian Antimicrobial Peptide Uperin 3.5 is a Cross-α/Cross-β Chameleon Functional Amyloid. PNAS 118 (3) e2014442118; 2021

Most Microorganisms form sessile multi-cellular biofilms in which they are protected against stress by a gel-like extracellular matrix composed of proteinaceous fibrils, various extracellular polysaccharides (EPS) and often DNA. Means to suppress or resolve biofilms are urgently needed given the looming health-crisis due to antibiotic-resistant bacterial strains. Our model organism Bacillus subtilis has disease-causing relatives such B. cereus and B. anthracis, and thus provides a means to investigate common biofilm formation mechanisms. In many cases, the major biofilm-associated protein forms amyloid-like fibrils. The situation is found different for Bacillus subtilis biofilms that contain filaments composed of b-sandwich do- mains that cannot be stained by Thioflavin T or congored. Solid-state NMR experiments re- vealed insertion of an initially flexible N-terminal TasA peptide segment into subsequent pro- tomers and further a whole set of restraints that is in agreement with a mildly helical filament, as predicted by AlphaFold. We further identified the mechanism by which the specific chaper- one TapA initiates filament growth. We present the three-dimensional structure of TapA and uncover the mechanism of TapA-supported growth of TasA filaments. In this context, we demonstrate unfolding of TasA monomers by TapA. A combination of analytical ultra-centrifu- gation and NMR reveals further a TapA concentration-dependent support of TasA oligomer formation, yet TapA is not consumed in this process. Instead, TapA serves as a template for TasA refolding and oligomerization by presenting its N-terminus to unfolded TasA as appearing outside the cell via the SecA pathway. In effect, a TasA filament structure is produced that consists of complemented V-set Ig folds, highly homologues to type I pili. Such pilus-homolo- gous structures were thus observed for the first time in Gram-positive bacteria. Based on our results, an analysis of conservation pattern enables the prediction of biofilm compatibility within Bacillaceae.

In contexts ranging from embryonic development to bacterial ecology, cell populations migrate chemotactically along self-generated chemical gradients, often forming a propagating front. I will theoretically show that the stability of such chemotactic fronts to morphological perturbations is determined by limitations in the ability of individual cells to sense and respond to the chemical gradient. Specifically, I will argue that cells at bulging parts of a front are exposed to a smaller gradient, which slows them down and promotes stability, but they also respond more strongly to the gradient, which speeds them up and promotes instability. We predict that this competition leads to chemotactic fingering when sensing is limited at too low chemical concentrations. Guided by this finding and by experimental data on E. coli chemotaxis, we suggest that the cells’ sensory machinery might have evolved to avoid these limitations and ensure stable front propagation. Finally, as sensing of any stimuli is necessarily limited, the principle of sensing-induced stability may operate in other types of directed migration such as durotaxis, electrotaxis, and phototaxis.

On solid surfaces, undomesticated strains of B. subtilis form intricately structured colonies, in which phenotypically different cell types are spatiotemporally coordinated to form robust microbial tissues and fruiting bodies. These structures provide a multicellular form for controlling population expansion, balancing growth with survival, and ultimately culminating in the formation of dormant endospores. In the context of such multicellular differentiation, antimicrobial compounds can play a role in intraspecies competition, resulting in the death of a sub-population of genetically identical siblings for the benefit of the population. Cannibalism is a unique bacterial form of programmed cell death that links multicellular differentiation to endospore formation in Bacillus subtilis. Cannibalism was originally associated with the production of two toxins, the sporulation killing factor SKF and the sporulation delay protein SDP, which both target the cell envelope of B. subtilis [1]. More recently, we have described a comparable role for a third antimicrobial compound, the epipeptide EPE, which is produced by the gene products of the epeXEPAB operon [2, 3]. EPE targets the cell envelope of B. subtilis and results in severe perturbations of the cytoplasmic membrane, including dissipating the membrane potential via membrane permeabilization, accompanied by a rapid reduction of membrane fluidity and lipid domain formation. Alterations of the balance between autoimmunity and production of both EPE and SDP result in dramatic changes in differentiation and hence colony morphology. Taken together, cannibalism seems to represent a central checkpoint in tissue formation, which (i) provides resources to either delay or ultimately fuel sporulation, (ii) structures and functionalizes the colonies, and might ultimately serve to (iii) balance the ratio of different cell types in structured multicellular populations. [1] Höfler et al. (2016), Microbiology 162(1):164-176. [2] Popp et al. (2020) Front Microbiol. 11:151. [3] Popp et al. (2021) Microb Physiol. 31(3):306-318.