Multicellular Communities Research Articles

Architectural dissection of adhesive bacterial cell surface appendages from a "molecular machines" viewpoint.

The ability of bacteria to interact with and respond to their environment is crucial to their lifestyle and survival. Bacterial cells routinely need to engage with extracellular target molecules, in locations spatially separated from their cell surface. Engagement with distant targets allows bacteria to adhere to abiotic surfaces and host cells, sense harmful or friendly molecules in their vicinity, as well as establish symbiotic interactions with neighboring cells in multicellular communities such as biofilms. Binding to extracellular molecules also facilitates transmission of information back to the originating cell, allowing the cell to respond appropriately to external stimuli, which is critical throughout the bacterial life cycle. This requirement of bacteria to bind to spatially separated targets is fulfilled by a myriad of specialized cell surface molecules, which often have an extended, filamentous arrangement. In this review, we compare and contrast such molecules from diverse bacteria, which fulfil a range of binding functions critical for the cell. Our comparison shows that even though these extended molecules have vastly different sequence, biochemical and functional characteristics, they share common architectural principles that underpin bacterial adhesion in a variety of contexts. In this light, we can consider different bacterial adhesins under one umbrella, specifically from the point of view of a modular molecular machine, with each part fulfilling a distinct architectural role. Such a treatise provides an opportunity to discover fundamental molecular principles governing surface sensing, bacterial adhesion, and biofilm formation.

RpoS activates formation of Salmonella Typhi biofilms and drives persistence in the gall bladder.

The development of strategies for targeting the asymptomatic carriage of Salmonella Typhi in chronic typhoid patients has suffered owing to our basic lack of understanding of the molecular mechanisms that enable the formation of S. Typhi biofilms. Traditionally, studies have relied on cholesterol-attached biofilms formed by a closely related serovar, Typhimurium, to mimic multicellular Typhi communities formed on human gallstones. In long-term infections, S. Typhi adopts the biofilm lifestyle to persist in vivo and survive in the carrier state, ultimately leading to the spread of infections via the fecal-oral route of transmission. In the present work, we studied S. Typhi biofilms directly, applied targeted as well as genome-wide genetic approaches to uncover unique biofilm components that do not conform to the CsgD-dependent pathway established in S. Typhimurium. We undertook a genome-wide Tn5 mutation screen in H58, a clinically relevant multidrug resistance strain of S. Typhi, in gallstone-mimicking conditions. We generated New Generation Sequencing libraries based on the ClickSeq technology to identify the key regulators, IraP and RpoS, and the matrix components Sth fimbriae, Vi capsule and lipopolysaccharide. We discovered that the starvation sigma factor, RpoS, was required for the transcriptional activation of matrix-encoding genes in vitro, and for S. Typhi colonization in persistent infections in vivo, using a heterologous fish larval model. An rpoS null mutant failed to colonize the gall bladder in chronic zebrafish infections. Overall, our work uncovered a novel RpoS-driven, CsgD-independent paradigm for the formation of cholesterol-attached Typhi biofilms, and emphasized the role(s) of stress signaling pathways for adaptation in chronic infections. Our identification of the biofilm regulators in S. Typhi paves the way for the development of drugs against typhoid carriage, which will ultimately control the increased incidence of gall bladder cancer in typhoid carriers.

Single-cell RNA sequencing reveals intratumoral heterogeneity and multicellular community in primary hepatocellular carcinoma underlying microvascular invasion

BackgroundMicrovascular invasion (MVI) is associated with an unfavorable prognosis and early recurrence of hepatocellular carcinoma (HCC), which is the crucial pathological hallmark of immunotherapy. While microvascular invasion (MVI) in hepatocellular carcinoma (HCC) currently lacks a detailed single-cell analysis of the tumor microenvironment (TME), it holds significant promise for immunotherapy using immune checkpoint inhibitors (ICI). MethodsWe performed single-cell RNA sequencing (scRNA-seq) on 3 MVI positive (MVIP) and 14 MVI-negative (MVIN) tumor tissues, as well as their paired adjacent non-tumoral tissues. ResultsWe identified SPP1+ macrophages and CD4+ proliferative T cells as intertumoral populations critical for the formation of cold tumors and immunosuppressive environments in MVI-positive patients and verified their prognostic value in correlation with MVIP HCC patients. Additionally, we identified SPP1+ dominated interactions between SPP1+ macrophages and the immunosuppressive T population as contributors to MVI destruction and tumorigenesis. ConclusionsWe provide a comprehensive single-cell atlas of HCC patients with MVI, shedding light on the immunosuppressive ecosystem and upregulated signaling associated with MVI. These findings demonstrate that intercellular mechanisms drive MVI and provide a potential immunotherapeutic target for HCC patients with HCC and underlying MVI.

Combinatorial control of Pseudomonas aeruginosa biofilm development by quorum-sensing and nutrient-sensing regulators.

The human pathogen Pseudomonas aeruginosa, a leading cause of hospital-acquired infections, inhabits and forms sessile antibiotic-resistant communities called biofilms in a wide range of biotic and abiotic environments. In this study, we examined how two global sensory signaling pathways-the RhlR quorum-sensing system and the CbrA/CbrB nutritional adaptation system-intersect to control biofilm development. Previous work has shown that individually these two systems repress biofilm formation. Here, we used biofilm analyses, RNA-seq, and reporter assays to explore the combined effect of information flow through RhlR and CbrA on biofilm development. We find that the ΔrhlRΔcbrA double mutant exhibits a biofilm morphology and an associated transcriptional response distinct from wildtype and the parent ΔrhlR and ΔcbrA mutants indicating codominance of each signaling pathway. The ΔrhlRΔcbrA mutant gains suppressor mutations that allow biofilm expansion; these mutations map to the crc gene resulting in loss of function of the carbon catabolite repression protein Crc. Furthermore, the combined absence of RhlR and CbrA leads to a drastic reduction in the abundance of the Crc antagonist small RNA CrcZ. Thus, CrcZ acts as the molecular convergence point for quorum- and nutrient-sensing cues. We find that in the absence of antagonism by CrcZ, Crc promotes the expression of biofilm matrix components-Pel exopolysaccharide, and CupB and CupC fimbriae. Therefore, this study uncovers a regulatory link between nutritional adaption and quorum sensing with potential implications for anti-biofilm targeting strategies.IMPORTANCEBacteria often form multicellular communities encased in an extracytoplasmic matrix called biofilms. Biofilm development is controlled by various environmental stimuli that are decoded and converted into appropriate cellular responses. To understand how information from two distinct stimuli is integrated, we used biofilm formation in the human pathogen Pseudomonas aeruginosa as a model and studied the intersection of two global sensory signaling pathways-quorum sensing and nutritional adaptation. Global transcriptomics on biofilm cells and reporter assays suggest parallel regulation of biofilms by each pathway that converges on the abundance of a small RNA antagonist of the carbon catabolite repression protein, Crc. We find a new role of Crc as it modulates the expression of biofilm matrix components in response to the environment. These results expand our understanding of the genetic regulatory strategies that allow P. aeruginosa to successfully develop biofilm communities.

A biofilm-tropic Pseudomonas aeruginosa bacteriophage uses the exopolysaccharide Psl as receptor.

Bacteria in nature can exist in multicellular communities called biofilms. Biofilms also form in the course of many infections. Pseudomonas aeruginosa infections frequently involve biofilms, which contribute materially to the difficulty to treat these infections with antibiotic therapy. Many biofilm-related characteristics are controlled by the second messenger, cyclic-di-GMP, which is upregulated on surface contact. Among these factors is the exopolysaccharide Psl, which is a critically important component of the biofilm matrix. Here we describe the discovery of a P. aeruginosa bacteriophage, which we have called Clew-1, that directly binds to and uses Psl as a receptor. While this phage does not efficiently infect planktonically growing bacteria, it can disrupt P. aeruginosa biofilms and replicate in biofilm bacteria. We further demonstrate that the Clew-1 can reduce the bacterial burden in a mouse model of P. aeruginosa keratitis, which is characterized by the formation of a biofilm on the cornea. Due to its reliance on Psl for infection, Clew-1 does not actually form plaques on wild-type bacteria under standard in vitro conditions. This argues that our standard isolation procedures likely exclude bacteriophage that are adapted to using biofilm markers for infection. Importantly, the manner in which we isolated Clew-1 can be easily extended to other strains of P. aeruginosa and indeed other bacterial species, which will fuel the discovery of other biofilm-tropic bacteriophage and expand their therapeutic use.

Biofilm dispersal patterns revealed using far-red fluorogenic probes.

Bacteria frequently colonize niches by forming multicellular communities called biofilms. To explore new territories, cells exit biofilms through an active process called dispersal. Biofilm dispersal is essential for bacteria to spread between infection sites, yet how the process is executed at the single-cell level remains mysterious. Here, we characterize dispersal at unprecedented resolution for the global pathogen Vibrio cholerae. To do so, we first developed a far-red cell-labeling strategy that overcomes pitfalls of fluorescent protein-based approaches. We reveal that dispersal initiates at the biofilm periphery and ~25% of cells never disperse. We define novel micro-scale patterns that occur during dispersal, including biofilm compression and the formation of dynamic channels. These patterns are attenuated in mutants that reduce overall dispersal or that increase dispersal at the cost of homogenizing local mechanical properties. Collectively, our findings provide fundamental insights into the mechanisms of biofilm dispersal, advancing our understanding of how pathogens disseminate.

Bacterial single-cell RNA sequencing captures biofilm transcriptional heterogeneity and differential responses to immune pressure.

Biofilm formation is an important mechanism of survival and persistence for many bacterial pathogens. These multicellular communities contain subpopulations of cells that display vast metabolic and transcriptional diversity along with high recalcitrance to antibiotics and host immune defenses. Investigating the complex heterogeneity within biofilm has been hindered by the lack of a sensitive and high-throughput method to assess stochastic transcriptional activity and regulation between bacterial subpopulations, which requires single-cell resolution. We have developed an optimized bacterial single-cell RNA sequencing method, BaSSSh-seq, to study Staphylococcus aureus diversity during biofilm growth and transcriptional adaptations following immune cell exposure. We validated the ability of BaSSSh-seq to capture extensive transcriptional heterogeneity during biofilm compared to planktonic growth. Application of new computational tools revealed transcriptional regulatory networks across the heterogeneous biofilm subpopulations and identification of gene sets that were associated with a trajectory from planktonic to biofilm growth. BaSSSh-seq also detected alterations in biofilm metabolism, stress response, and virulence that were tailored to distinct immune cell populations. This work provides an innovative platform to explore biofilm dynamics at single-cell resolution, unlocking the potential for identifying biofilm adaptations to environmental signals and immune pressure.

WebCM: A Web-Based Platform for Multiuser Individual-Based Modeling of Multicellular Microbial Populations and Communities.

WebCM is a web platform that enables users to create, edit, run, and view individual-based simulations of multicellular microbial populations and communities on a remote compute server. WebCM builds upon the simulation software CellModeller in the back end and provides users with a web-browser-based modeling interface including model editing, execution, and playback. Multiple users can run and manage multiple simulations simultaneously, sharing the host hardware. Since it is based on CellModeller, it can utilize both GPU and CPU parallelization. The user interface provides real-time interactive 3D graphical representations for inspection of simulations at all time points, and the results can be downloaded for detailed offline analysis. It can be run on cloud computing services or on a local server, allowing collaboration within and between laboratories.

Escherichia coli self-organizes developmental rosettes

Rosettes are self-organizing, circular multicellular communities that initiate developmental processes, like organogenesis and embryogenesis, in complex organisms. Their formation results from the active repositioning of adhered sister cells and is thought to distinguish multicellular organisms from unicellular ones. Though common in eukaryotes, this multicellular behavior has not been reported in bacteria. In this study, we found that Escherichia coli forms rosettes by active sister-cell repositioning. After division, sister cells "fold" to actively align at the 2- and 4-cell stages of clonal division, thereby producing rosettes with characteristic quatrefoil configuration. Analysis revealed that folding follows an angular random walk, composed of ~1 µm strokes and directional randomization. We further showed that this motion was produced by the flagellum, the extracellular tail whose rotation generates swimming motility. Rosette formation was found to require de novo flagella synthesis suggesting it must balance the opposing forces of Ag43 adhesion and flagellar propulsion. We went on to show that proper rosette formation was required for subsequent morphogenesis of multicellular chains, rpoS gene expression, and formation of hydrostatic clonal-chain biofilms. Moreover, we found self-folding rosette-like communities in the standard motility assay, indicating that this behavior may be a general response to hydrostatic environments in E. coli. These findings establish self-organization of clonal rosettes by a prokaryote and have implications for evolutionary biology, synthetic biology, and medical microbiology.

DNA Reaction Circuits to Establish Designated Biological Functions in Multicellular Community.

In multicellular organisms, individual cells are coordinated through complex communication networks to accomplish various physiological tasks. Aiming to establish new biological functions in the multicellular community, we used DNA as the building block to develop a cascade of nongenetic reaction circuits to establish a dynamic cell-cell communication network. Utilizing membrane-anchored amphiphilic DNA tetrahedra (TDN) as the nanoscaffold, reaction circuits were incorporated into three unrelated cells in order to uniquely regulate their sense-and-response behaviors. As a proof-of-concept, this step enabled these cells to simulate significant biological events involved in T cell-mediated anticancer immunity. Such events included cancer-associated antigen recognition and the presentation of antigen-presenting cells (APCs), APC-facilitated T cell activation and dissociation, and T cell-mediated cancer targeting and killing. By combining the excellent programmability and molecular recognition ability of DNA, our cell-surface reaction circuits hold promise for mimicking and manipulating many biological processes.

Formation of recurring transient Ca2+-based intercellular communities during Drosophila hematopoiesis

Tissue development occurs through a complex interplay between many individual cells. Yet, the fundamental question of how collective tissue behavior emerges from heterogeneous and noisy information processing and transfer at the single-cell level remains unknown. Here, we reveal that tissue scale signaling regulation can arise from local gap-junction mediated cell-cell signaling through the spatiotemporal establishment of an intermediate-scale of transient multicellular communication communities over the course of tissue development. We demonstrated this intermediate scale of emergent signaling using Ca2+ signaling in the intact, ex vivo cultured, live developing Drosophila hematopoietic organ, the lymph gland. Recurrent activation of these transient signaling communities defined self-organized signaling "hotspots" that gradually formed over the course of larva development. These hotspots receive and transmit information to facilitate repetitive interactions with nonhotspot neighbors. Overall, this work bridges the scales between single-cell and emergent group behavior providing key mechanistic insight into how cells establish tissue-scale communication networks.

Abstract 6846: Tumor cell metaplastic programs and failure of T cell remodeling drive resistance and progression to frontline chemoimmunotherapy in advanced gastric cancer

Abstract Adding aPD1 to 5-FU/platinum in advanced gastroesophageal adenocarcinomas (GEA) has yielded modest and heterogeneous results. Understanding cooperativity between these two treatment modalities will inform novel combination treatments for GEA. Towards this end, we conducted a trial (n = 47) sequentially adding pembrolizumab to 5-FU/platinum in previously untreated advanced GEA. Using serial biopsy of the primary tumor at baseline, after one cycle of 5-FU/platinum, and after the addition of pembrolizumab we transcriptionally profiled 358,067 single cells and obtained TCR-sequences from single tumor-resident T cells. Stratifying patients into slow- (> 6 months) and fast-progressing (< 6 months) groups based on progression-free survival allowed us to quantify differences in immune, stromal and epithelial subpopulations across treatment time points. Using topic modeling with consensus non-negative matrix factorization (cNMF), we identified gene expression programs in each population at each time point and developed a methodology to verify the stability of those programs across the cohort using cross-validation. Faster clinical progression featured decreased M1-like macrophage repolarization and increased MUC5A and MSLN containing metaplasia programs in tumor cells. We next selected features from cell state gene programs and identified trajectories of tumor cells across a spectrum of treatment sensitive and resistant states. We identified several factors that correlated with these trajectories and regulons that may be influencing them. After pembrolizumab we observed slower progression was associated with further increased CD8 T cell infiltration with increased CXCL13+ T cells. We next calculated covarying programs between patient samples at each time point and performed permutation testing to identify which programs (and respective cell types they were expressed in) were significantly correlated to infer the presence of multicellular tumor communities. We found that the tumor cell interferon program co-varied with the CXCL13 T cell program, and the emergence of a covarying module with tumor-reactive CXCL13 program expression happens as early as after the first dose of chemotherapy. To better understand the behavior of tumor reactive T cells within the tumor microenvironment, we leveraged TCR-sequencing data to study T cell clonality and diversity across treatment time points and between fast and slow progressors. Our data provide an invaluable resource to dissect chemoimmunotherapy response in tumor cells, understand T cell behavior and propose strategies to drive anti-tumor immune hub formation to expand the portion of patients benefiting from immunotherapy approaches. Citation Format: Samuel J. Wright, Minae An, Byung Hoon Min, You Jeong Heo, Milan Parikh, Lynn Bi, Hyuk Lee, Taejun Kim, Song-Yi Lee, Jeonghyeon Moon, Ryan J. Park, Matthew R. Strickland, Woong Yang Park, Won Ki Kang, Kyoung-Mee Kim, Seung Tae Kim, Arnav Mehta, Samuel J. Klempner, Jeeyun Lee. Tumor cell metaplastic programs and failure of T cell remodeling drive resistance and progression to frontline chemoimmunotherapy in advanced gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6846.

Glycerol metabolism impacts biofilm phenotypes and virulence in Pseudomonas aeruginosa via the Entner-Doudoroff pathway.

Pseudomonas aeruginosa is a ubiquitous bacterium and a notorious opportunistic pathogen that forms biofilm structures in response to many environmental cues. Biofilm formation includes attachment to surfaces and the production of the exopolysaccharide Pel, which is present in both the PAO1 and PA14 laboratory strains of P. aeruginosa. Biofilms help protect bacterial cells from host defenses and antibiotics and abet infection. The carbon source used by the cells also influences biofilm, but these effects have not been deeply studied. We show here that glycerol, which can be liberated from host surfactants during infection, encourages surface attachment and magnifies colony morphology differences. We find that glycerol kinase is important but not essential for glycerol utilization and relatively unimportant for biofilm behaviors. Among downstream enzymes predicted to take part in glycerol utilization, Edd stood out as being important for glycerol utilization and for enhanced biofilm phenotypes in the presence of glycerol. Thus, gluconeogenesis and catabolism of anabolically produced glucose appear to impact not only the utilization of glycerol but also glycerol-stimulated biofilm phenotypes. Finally, waxworm moth larvae and nematode infection models reveal that interruption of the Entner-Doudoroff pathway, but not abrogation of glycerol phosphorylation, unexpectedly increases P. aeruginosa lethality in both acute and chronic infections, even while stimulating a stronger immune response by Caenorhabditis elegans.IMPORTANCEPseudomonas aeruginosa, the ubiquitous environmental bacterium and human pathogen, forms multicellular communities known as biofilms in response to various stimuli. We find that glycerol, a common carbon source that bacteria can use for energy and biosynthesis, encourages biofilm behaviors such as surface attachment and colony wrinkling by P. aeruginosa. Glycerol can be derived from surfactants that are present in the human lungs, a common infection site. Glycerol-stimulated biofilm phenotypes do not depend on phosphorylation of glycerol but are surprisingly impacted by a glucose breakdown pathway, suggesting that it is glycerol utilization, and not its mere presence or cellular import, that stimulates biofilm phenotypes. Moreover, the same mutations that block glycerol-stimulated biofilm phenotypes also impact P. aeruginosa virulence in both acute and chronic animal models. Notably, a glucose-breakdown mutant (Δedd) counteracts biofilm phenotypes but shows enhanced virulence and stimulates a stronger immune response in Caenorhabditis elegans.

Bacillus subtilis Matrix Protein TasA is Interfacially Active, but BslA Dominates Interfacial Film Properties.

Microbial growth often occurs within multicellular communities called biofilms, where cells are enveloped by a protective extracellular matrix. Bacillus subtilis serves as a model organism for biofilm research and produces two crucial secreted proteins, BslA and TasA, vital for biofilm matrix formation. BslA exhibits surface-active properties, spontaneously self-assembling at hydrophobic/hydrophilic interfaces to form an elastic protein film, which renders B. subtilis biofilm surfaces water-repellent. TasA is traditionally considered a fiber-forming protein with multiple matrix-related functions. In our current study, we investigate whether TasA also possesses interfacial properties and whether it has any impact on BslA's ability to form an interfacial protein film. Our research demonstrates that TasA indeed exhibits interfacial activity, partitioning to hydrophobic/hydrophilic interfaces, stabilizing emulsions, and forming an interfacial protein film. Interestingly, TasA undergoes interface-induced restructuring similar to BslA, showing an increase in β-strand secondary structure. Unlike BslA, TasA rapidly reaches the interface and forms nonelastic films that rapidly relax under pressure. Through mixed protein pendant drop experiments, we assess the influence of TasA on BslA film formation, revealing that TasA and other surface-active molecules can compete for interface space, potentially preventing BslA from forming a stable elastic film. This raises a critical question: how does BslA self-assemble to form the hydrophobic "raincoat" observed in biofilms in the presence of other potentially surface-active species? We propose a model wherein surface-active molecules, including TasA, initially compete with BslA for interface space. However, under lateral compression or pressure, BslA retains its position, expelling other molecules into the bulk. This resilience at the interface may result from structural rearrangements and lateral interactions between BslA subunits. This combined mechanism likely explains BslA's role in forming a stable film integral to B. subtilis biofilm hydrophobicity.

Cell competition: emerging signaling and unsolved questions.

Multicellular communities have an intrinsic mechanism that optimizes their structure and function via cell-cell communication. One of the driving forces for such self-organization of the multicellular system is cell competition, the elimination of viable unfit or deleterious cells via cell-cell interaction. Studies in Drosophila and mammals have identified multiple mechanisms of cell competition caused by different types of mutations or cellular changes. Intriguingly, recent studies have found that different types of "losers" of cell competition commonly show reduced protein synthesis. In Drosophila, the reduction in protein synthesis levels in loser cells is caused by phosphorylation of the translation initiation factor eIF2α via a bZip transcription factor Xrp1. Given that a variety of cellular stresses converge on eIF2α phosphorylation and thus global inhibition of protein synthesis, cell competition may be a machinery that optimizes multicellular fitness by removing stressed cells. In this review, we summarize and discuss emerging signaling mechanisms and critical unsolved questions, as well as the role of protein synthesis in cell competition.

Vibrio fischeri: a model for host-associated biofilm formation.

Multicellular communities of adherent bacteria known as biofilms are often detrimental in the context of a human host, making it important to study their formation and dispersal, especially in animal models. One such model is the symbiosis between the squid Euprymna scolopes and the bacterium Vibrio fischeri. Juvenile squid hatch aposymbiotically and selectively acquire their symbiont from natural seawater containing diverse environmental microbes. Successful pairing is facilitated by ciliary movements that direct bacteria to quiet zones on the surface of the squid's symbiotic light organ where V. fischeri forms a small aggregate or biofilm. Subsequently, the bacteria disperse from that aggregate to enter the organ, ultimately reaching and colonizing deep crypt spaces. Although transient, aggregate formation is critical for optimal colonization and is tightly controlled. In vitro studies have identified a variety of polysaccharides and proteins that comprise the extracellular matrix. Some of the most well-characterized matrix factors include the symbiosis polysaccharide (SYP), cellulose polysaccharide, and LapV adhesin. In this review, we discuss these components, their regulation, and other less understood V. fischeri biofilm contributors. We also highlight what is currently known about dispersal from these aggregates and host cues that may promote it. Finally, we briefly describe discoveries gleaned from the study of other V. fischeri isolates. By unraveling the complexities involved in V. fischeri's control over matrix components, we may begin to understand how the host environment triggers transient biofilm formation and dispersal to promote this unique symbiotic relationship.

Fitness effects of a demography-dispersal trade-off in expandingSaccharomyces cerevisiaemats.

Fungi expand in space and time to form complex multicellular communities. The mechanisms by which they do so can vary dramatically and determine the life-history and dispersal traits of expanding populations. These traits influence deterministic and stochastic components of evolution, resulting in complex eco-evolutionary dynamics during colony expansion. We perform experiments on budding yeast strains genetically engineered to display rough-surface and smooth-surface phenotypes in colony-like structures called 'mats'. Previously, it was shown that the rough-surface strain has a competitive advantage over the smooth-surface strain when grown on semi-solid media. We experimentally observe the emergence and expansion of segments with a distinct smooth-surface phenotype during rough-surface mat development. We propose a trade-off between dispersal and local carrying capacity to explain the relative fitness of these two phenotypes. Using a modified stepping-stone model, we demonstrate that this trade-off gives the high-dispersing, rough-surface phenotype a competitive advantage from standing variation, but that it inhibits this phenotype's ability to invade a resident smooth-surface population via mutation. However, the trade-off improves the ability of the smooth-surface phenotype to invade in rough-surface mats, replicating the frequent emergence of smooth-surface segments in experiments. Together, these computational and experimental findings advance our understanding of the complex eco-evolutionary dynamics of fungal mat expansion.

Residual cells and nutrient availability guide wound healing in bacterial biofilms.

Biofilms are multicellular heterogeneous bacterial communities characterized by social-like division of labor, and remarkable robustness with respect to external stresses. Increasingly often an analogy between biofilms and arguably more complex eukaryotic tissues is being drawn. One illustrative example of where this analogy can be practically useful is the process of wound healing. While it has been extensively studied in eukaryotic tissues, the mechanism of wound healing in biofilms is virtually unexplored. Combining experiments in Bacillus subtilis bacteria, a model organism for biofilm formation, and a lattice-based theoretical model of biofilm growth, we studied how biofilms recover after macroscopic damage. We suggest that nutrient gradients and the abundance of proliferating cells are key factors augmenting wound closure. Accordingly, in the model, cell quiescence, nutrient fluxes, and biomass represented by cells and self-secreted extracellular matrix are necessary to qualitatively recapitulate the experimental results for damage repair. One of the surprising experimental findings is that residual cells, persisting in a damaged area after removal of a part of the biofilm, prominently affect the healing process. Taken together, our results outline the important roles of nutrient gradients and residual cells on biomass regrowth on macroscopic scales of the whole biofilm. The proposed combined experiment-simulation framework opens the way to further investigate the possible relation between wound healing, cell signaling and cell phenotype alternation in the local microenvironment of the wound.

Microfluidic approaches in microbial ecology.

Microbial life is at the heart of many diverse environments and regulates most natural processes, from the functioning of animal organs to the cycling of global carbon. Yet, the study of microbial ecology is often limited by challenges in visualizing microbial processes and replicating the environmental conditions under which they unfold. Microfluidics operates at the characteristic scale at which microorganisms live and perform their functions, thus allowing for the observation and quantification of behaviors such as growth, motility, and responses to external cues, often with greater detail than classical techniques. By enabling a high degree of control in space and time of environmental conditions such as nutrient gradients, pH levels, and fluid flow patterns, microfluidics further provides the opportunity to study microbial processes in conditions that mimic the natural settings harboring microbial life. In this review, we describe how recent applications of microfluidic systems to microbial ecology have enriched our understanding of microbial life and microbial communities. We highlight discoveries enabled by microfluidic approaches ranging from single-cell behaviors to the functioning of multi-cellular communities, and we indicate potential future opportunities to use microfluidics to further advance our understanding of microbial processes and their implications.

How Do Phages Disrupt the Structure of Enterococcus faecalis Biofilm?

Biofilms are composed of multicellular communities of microbial cells and their self-secreted extracellular polymeric substances (EPS). The viruses named bacteriophages can infect and lyze bacterial cells, leading to efficient biofilm eradication. The aim of this study was to analyze how bacteriophages disrupt the biofilm structure by killing bacterial cells and/or by damaging extracellular polysaccharides, proteins, and DNA. The use of colorimetric and spectrofluorimetric methods and confocal laser scanning microscopy (CLSM) enabled a comprehensive assessment of phage activity against E. faecalis biofilms. The impact of the phages vB_Efa29212_2e and vB_Efa29212_3e was investigated. They were applied separately or in combination on 1-day and 7-day-old biofilms. Phages 2e effectively inhibited the growth of planktonic cells with a limited effect on the biofilm. They did not notably affect extracellular polysaccharides and proteins; however, they increased DNA levels. Phages 3e demonstrated a potent and dispersing impact on E. faecalis biofilms, despite being slightly less effective than bacteriophages 2e against planktonic cells. Phages 3e reduced the amount of extracellular polysaccharides and increased eDNA levels in both 1-day-old and 7-day-old biofilm cultures. Phage cocktails had a strong antimicrobial effect on both planktonic and biofilm-associated bacteria. A significant reduction in the levels of polysaccharides, proteins, and eDNA in 1-day-old biofilm samples was noted, which confirms that phages interfere with the structure of E. faecalis biofilm by killing bacterial cells and affecting extracellular polymer levels.