Multicellular Communities Research Articles

Single-cell dissection of the multicellular ecosystem and molecular features underlying microvascular invasion in HCC.

Microvascular invasion (MVI) is a crucial pathological hallmark of HCC that is closely associated with poor outcomes, early recurrence, and intrahepatic metastasis following surgical resection and transplantation. However, the intricate tumor microenvironment and transcriptional programs underlying MVI in HCC remain poorly understood. We performed single-cell RNA sequencing of 46,789 individual cells from 10 samples of MVI+ (MVI present) and MVI- (MVI absent) patients with HCC. We conducted comprehensive and comparative analyses to characterize cellular and molecular features associated with MVI and validated key findings using external bulk, single-cell, and spatial transcriptomic datasets coupled with multiplex immunofluorescence assays. The comparison identified specific subtypes of immune and stromal cells critical to the formation of the immunosuppressive and pro-metastatic microenvironment in MVI+ tumors, including cycling T cells, lysosomal associated membrane protein 3+ dendritic cells, triggering receptor expressed on myeloid cells 2+ macrophages, myofibroblasts, and arterial i endothelial cells. MVI+ malignant cells are characterized by high proliferation rates, whereas MVI- malignant cells exhibit an inflammatory milieu. Additionally, we identified the midkine-dominated interaction between triggering receptor expressed on myeloid cells 2+ macrophages and malignant cells as a contributor to MVI formation and tumor progression. Notably, we unveiled a spatially co-located multicellular community exerting a dominant role in shaping the immunosuppressive microenvironment of MVI and correlating with unfavorable prognosis. This study provides a comprehensive single-cell atlas of MVI in HCC, shedding light on the complex multicellular ecosystem and molecular features associated with MVI. These findings deepen our understanding of the underlying mechanisms driving MVI and provide valuable insights for improving clinical diagnosis and developing more effective treatment strategies.

RecA levels modulate biofilm development in Acinetobacter baumannii.

Infections caused by Acinetobacter baumannii, a Gram-negative opportunistic pathogen, are difficult to eradicate due to the bacterium's propensity to quickly gain antibiotic resistances and form biofilms, a protective bacterial multicellular community. The A. baumannii DNA damage response (DDR) mediates the antibiotic resistance acquisition and regulates RecA in an atypical fashion; both RecALow and RecAHigh cell types are formed in response to DNA damage. The findings of this study demonstrate that the levels of RecA can influence formation and dispersal of biofilms. RecA loss results in surface attachment and prominent biofilms, while elevated RecA leads to diminished attachment and dispersal. These findings suggest that the challenge to treat A. baumannii infections may be explained by the induction of the DDR, common during infection, as well as the delicate balance between maintaining biofilms in low RecA cells and promoting mutagenesis and dispersal in high RecA cells. This study underscores the importance of understanding the fundamental biology of bacteria to develop more effective treatments for infections.

Coordinated proteome change precedes cell lysis and death in a mat-forming cyanobacterium

Cyanobacteria form dense multicellular communities that experience transient conditions in terms of access to light and oxygen. These systems are productive but also undergo substantial biomass turnover through cell death, supplementing heightened heterotrophic respiration. Here we use metagenomics and metaproteomics to survey the molecular response of a mat-forming cyanobacterium undergoing mass cell lysis after exposure to dark and anoxic conditions. A lack of evidence for viral, bacterial, or eukaryotic antagonism contradicts commonly held beliefs on the causative agent for cyanobacterial death during dense growth. Instead, proteogenomics data indicated that lysis likely resulted from a genetically programmed response triggered by a failure to maintain osmotic pressure in the wake of severe energy limitation. Cyanobacterial DNA was rapidly degraded, yet cyanobacterial proteins remained abundant. A subset of proteins, including enzymes involved in amino acid metabolism, peptidases, toxin-antitoxin systems, and a potentially self-targeting CRISPR-Cas system, were upregulated upon lysis, indicating possible involvement in the programmed cell death response. We propose this natural form of cell death could provide new pathways for controlling harmful algal blooms and for sustainable bioproduct production.

Cell behaviors underlying Myxococcus xanthus aggregate dispersal.

The soil bacterium Myxococcus xanthus is a model organism with a set of diverse behaviors. These behaviors include the starvation-induced multicellular development program, in which cells move collectively to assemble multicellular aggregates. After initial aggregates have formed, some will disperse, with smaller aggregates having a higher chance of dispersal. Initial aggregation is driven by two changes in cell behavior: cells slow down inside of aggregates and bias their motion by reversing direction less frequently when moving toward aggregates. However, the cell behaviors that drive dispersal are unknown. Here, we use fluorescent microscopy to quantify changes in cell behavior after initial aggregates have formed. We observe that after initial aggregate formation, cells adjust the bias in reversal timings by initiating reversals more rapidly when approaching unstable aggregates. Using agent-based modeling, we then show dispersal is predominantly generated by this change in bias, which is strong enough to overcome slowdown inside aggregates. Notably, the change in reversal bias is correlated with the nearest aggregate size, connecting cellular activity to previously observed correlations between aggregate size and fate. To determine if this connection is consistent across strains, we analyze a second M. xanthus strain with reduced levels of dispersal. We find that far fewer cells near smaller aggregates modified their bias. This implies that aggregate dispersal is under genetic control, providing a foundation for further investigations into the role it plays in the life cycle of M. xanthus.IMPORTANCEUnderstanding the processes behind bacterial biofilm formation, maintenance, and dispersal is essential for addressing their effects on health and ecology. Within these multicellular communities, various cues can trigger differentiation into distinct cell types, allowing cells to adapt to their specific local environment. The soil bacterium Myxococcus xanthus forms biofilms in response to starvation, marked by cells aggregating into mounds. Some aggregates persist as spore-filled fruiting bodies, while others disperse after initial formation for unknown reasons. Here, we use a combination of cell tracking analysis and computational simulations to identify behaviors at the cellular level that contribute to aggregate dispersal. Our results suggest that cells in aggregates actively determine whether to disperse or persist and undergo a transition to sporulation based on a self-produced cue related to the aggregate size. Identifying these cues is an important step in understanding and potentially manipulating bacterial cell-fate decisions.

Identification of In-Droplet Multicellular Communities by Light-Induced Combinatorial DNA Barcoding.

A microdroplet co-culture system is useful for the parallel assessment of numerous possible cell-cell interactions by generating isolated subcommunities from a pool of heterogeneous cells. However, the integration of single-cell sequencing into such analysis has been limited due to the lack of effective molecular identifiers for each in-droplet subcommunity. Herein, we present a strategy for generating in-droplet subcommunity identifiers using DNA-functionalized microparticles encapsulated within microdroplets. These microparticles serve as initial information carriers, where their combinations act as distinct identifiers for in-droplet subcommunity. Upon optical trigger, DNA barcoding molecules encoding the microparticle information are once released in the microdroplets and then tag cell membranes. The tagged DNA molecules then serve as a second information carrier readable by single-cell sequencing to reconstitute the community in silico in the single-cell RNA sequencing data space.

Archaeal type IV pili stabilize Haloferax volcanii biofilms in flow

Multicellular communities of contiguous cells attached to solid surfaces called biofilms represent a common microbial strategy to improve resilience in adverse environments.1,2,3 While bacterial biofilms have been under intense investigation, whether archaeal biofilms follow similar assembly rules remains unknown.4,5Haloferax volcanii is an extremely halophilic euryarchaeon that commonly colonizes salt crust surfaces. H.volcanii produces long and thin appendages called type IV pili (T4Ps). These play a role in surface attachment and biofilm formation in both archaea and bacteria. In this study, we employed biophysical experiments to identify the function of T4Ps in H.volcanii biofilm morphogenesis. H.volcanii expresses not one but six types of major pilin subunits that are predicted to compose T4Ps. Non-invasive imaging of T4Ps in live cells using interferometric scattering (iSCAT) microscopy reveals that piliation varies across mutants expressing single major pilin isoforms. T4Ps are necessary to secure attachment of single cells to surfaces, and the adhesive strength of pilin mutants correlates with their level of piliation. In flow, H.volcanii forms clonal biofilms that extend in three dimensions. Notably, the expression of PilA2, a single pilin isoform, is sufficient to maintain levels of piliation, surface attachment, and biofilm formation that are indistinguishable from the wild type. Furthermore, we discovered that fluid flow stabilizes biofilm integrity; as in the absence of flow, biofilms tend to lose cohesion and disperse in a density-dependent manner. Overall, our results demonstrate that T4P-surface and possibly T4P-T4P interactions promote biofilm formation and integrity and that flow is a key factor regulating archaeal biofilm formation.

Multicellular communities are perturbed in the aging human brain and Alzheimer's disease.

The role of different cell types and their interactions in Alzheimer's disease (AD) is a complex and open question. Here, we pursued this question by assembling a high-resolution cellular map of the aging frontal cortex using single-nucleus RNA sequencing of 24 individuals with a range of clinicopathologic characteristics. We used this map to infer the neocortical cellular architecture of 638 individuals profiled by bulk RNA sequencing, providing the sample size necessary for identifying statistically robust associations. We uncovered diverse cell populations associated with AD, including a somatostatin inhibitory neuronal subtype and oligodendroglial states. We further identified a network of multicellular communities, each composed of coordinated subpopulations of neuronal, glial and endothelial cells, and we found that two of these communities are altered in AD. Finally, we used mediation analyses to prioritize cellular changes that might contribute to cognitive decline. Thus, our deconstruction of the aging neocortex provides a roadmap for evaluating the cellular microenvironments underlying AD and dementia.

Microfluidics for Biofilm Studies

Biofilms are multicellular communities held together by a self-produced extracellular matrix and exhibit a set of properties that distinguish them from free-living bacteria. Biofilms are exposed to a variety of mechanical and chemical cues resulting from fluid motion and mass transport. Microfluidics provides the precise control of hydrodynamic and physicochemical microenvironments to study biofilms in general. In this review, we summarize the recent progress made in microfluidics-based biofilm research, including understanding the mechanism of bacterial adhesion and biofilm development, assessment of antifouling and antimicrobial properties, development of advanced in vitro infection models, and advancement in methods to characterize biofilms. Finally, we provide a perspective on the future direction of microfluidics-assisted biofilm research.

Upregulation of ica Operon Governs Biofilm Formation by a Coagulase-Negative Staphylococcus caprae.

Staphylococcus caprae is a Gram-positive, coagulase-negative staphylococci (CoNS), which appears as commensal in the skin, as well as a prevalent mastitis pathogen of goats. Occasionally, it is also associated with infections in humans. Biofilm formation has been identified as a putative virulence factor in S. caprae. Biofilms are multicellular communities protected by a self-produced extracellular matrix (ECM), which facilitates the resistance of bacterial cells to antimicrobial treatments. The ECM is constructed by exopolysaccharides, including the major exopolysaccharide-polysaccharide intercellular adhesion (PIA), regulated by the ica operon in Staphylococcus species. The aim of this study was to characterize the expression of the ica operon in relation to biofilm formation in S. caprae. Results showed that within a few hours of growth, S. caprae could adhere to polystyrene surfaces, start to accumulate, and form biofilm. Peak biofilm biomass and maturation were reached after 48 h, followed by a reduction in biomass after 72 h. Confocal laser scanning microscopy showed the expression of matrix-associated proteins and polysaccharides at various time points. The expression dynamics of the ica operon were investigated using real-time reverse transcriptase PCR (RT)-qPCR, which showed elevated expression during the early stages of biofilm formation and subsequent downregulation throughout the biofilm aging process. In conclusion, our results show that the ica operon is essential in regulating biofilm formation in S. caprae, similar to other Staphylococcus species. Furthermore, the robustness of the observed biofilm phenotype could account for the successful intramammary colonization and may explain disease persistence caused by this pathogenic bacterium.

Abstract 3116: Spatial single-cell atlas of stage III colorectal cancer

Abstract Background: Spatial heterogeneity and multicellular interactions within the tumor microenvironment drive tumor progression and response to therapy, yet, tumor molecular profiling often requires dissociation of cells, losing the spatial context. Spatial proteomic technologies enable capturing single-cell information with the spatial context allowing us to link cancer-causing mutations to outcomes in cell signaling, responses and survival that will aid in diagnosis, prognostication, and treatment of cancer. We aim to capture single cells in intact tissue sections of stage III colorectal tumors from 52 patients to spatially characterize cell types, interactions, and organization of the tumor microenvironment in relation to genetic alterations and clinical outcomes. Methods: Using Hyperion Imaging Mass Cytometry (IMC), we profiled 16 protein markers across 170 tissue regions, capturing molecular signatures of tissue architecture, tumor cells, and immune cells. We developed an analysis pipeline to quantify single-cell protein expression and identify cell phenotypes. We preserved spatial information to characterize the interactions between neighboring cells and identify multicellular communities. We also generated whole exome sequencing data, RNAseq data and histopathological images from sections of the same tissue blocks. We compared cellular and spatial features between tumors harboring prognostic genomic evens such as TP53 deletion and MSI and between patients stratified by clinical outcomes such as survival and recurrence. Results: We spatially profiled over 800,000 cells to identify 10 tumor, immune and stromal cell phenotypes, and validated identified cell types against matched histopathology and CIBERSORTx deconvoluted RNAseq. To quantify cell-cell interactions, we measured the distance between neighboring cells and performed neighborhood enrichment analysis. We characterized the multicellular organization of the tumor microenvironment to identify 7 consistent cell neighborhoods of up to 10 adjacent cells. We quantified p53 expression in individual tumor cells and found that TP53 deletion (all heterozygous) had no significant association with p53 expression or p53+ tumor cell abundance, suggesting spatial proteomics reveals additional information absent in traditional sequencing. We find that MSI is exclusive of TP53 deletion and that MSI positive tumors had few p53+ tumor cells, increased CD8+ T cell composition and B cells that were further away from p53+ tumor cells compared to MSI negative tumors. Conclusions: Using Hyperion IMC to acquire a spatial single-cell atlas of stage III colorectal cancer enabled us to characterize the tumor microenvironment by defining cell types, quantifying cell interactions, and identifying multicellular communities. These features enable the linkage of prognostic genetic alternations and clinical outcomes with changes in the tumor microenvironment. Citation Format: Andrew Su, Minh Tran, HoJoon Lee, Anuja Sathe, Xiangqi Bai, Richard Cruz, Lance Pflieger, Quan Nguyen, Hanlee P. Ji, Terence Rhodes. Spatial single-cell atlas of stage III colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3116.

Community-specific diffusion characteristics determine resistance of biofilms to oxidative stress

Biofilms are multicellular communities with a spatial structure. Different from single-cell scale diffusion in planktonic systems, the diffusion distance becomes the dimension of multicellular clusters in a biofilm. Such differences in diffusion behavior affect the tolerance and response to exogenous stress. Here, we found that at the same doses of exogenous hydrogen peroxide (H2O2), planktonic Escherichia coli were completely killed within twohours, whereas the biofilm resumed growth in sixhours by building a catalase barrier to block H2O2 penetration, despite the growth burden. Unexpectedly, when we changed the carbon source from glucose to glycerol, H2O2 instantly counterintuitively boosted biofilm growth due to supplemental oxygen, which was the growth-limiting factor. We further demonstrated that the energy metabolism modes determined the growth-limiting factor, which then determined the two patterns of biofilms resistances to H2O2.

Environmental DNA metabarcoding describes biodiversity across marine gradients

Abstract In response to climate change, biodiversity patterns in the oceans are predicted to shift rapidly, thus increasing the need for efficient monitoring methods. Environmental DNA (eDNA) metabarcoding recently emerged as a potent and cost-effective candidate to answer this challenge. We targeted three molecular markers to determine multicellular metazoan communities from two timepoints across a long-standing transect in the Southern Hemisphere, the Munida Observational Time Series. We detected four community types across the successive water masses—neritic, sub-tropical, frontal, and sub-Antarctic—crossed by the transect, together with important community differences between the two sampling points. From indicator species analysis, we found diversity patterns were mostly driven by planktonic organisms. Mesopelagic communities differed from surface-water communities in the sub-Antarctic water mass, with at-depth communities dominated by single-cellular organisms. We evaluate the ability of eDNA to detect species-compositional changes across surface and depth gradients and lay the foundations for using this technique in multi-trophic environmental monitoring efforts across long time series. We observed community differences across time and space. More intensive sampling will be critical to fully capture diversity across marine gradients, but this multi-trophic method represents an invaluable opportunity to understand shifts in marine biota.

Macroscopic quorum sensing sustains differentiating embryonic stem cells

Cells can secrete molecules that help each other’s replication. In cell cultures, chemical signals might diffuse only within a cell colony or between colonies. A chemical signal’s interaction length—how far apart interacting cells are—is often assumed to be some value without rigorous justifications because molecules’ invisible paths and complex multicellular geometries pose challenges. Here we present an approach, combining mathematical models and experiments, for determining a chemical signal’s interaction length. With murine embryonic stem (ES) cells as a testbed, we found that differentiating ES cells secrete FGF4, among others, to communicate over many millimeters in cell culture dishes and, thereby, form a spatially extended, macroscopic entity that grows only if its centimeter-scale population density is above a threshold value. With this ‘macroscopic quorum sensing’, an isolated macroscopic, but not isolated microscopic, colony can survive differentiation. Our integrated approach can determine chemical signals’ interaction lengths in generic multicellular communities.

Removing Biofilms in the Isthmus by Endodontic Surgery

Biofilms are multicellular communities which are composed of prokaryotic and/or eukaryotic cells embedded in matrix. More than 500 bacterial species have been identified in dental biofilms, which pose a harmful risk to dental health. Isthmus is a ribbon-shaped communication between two root canals which contains pulp tissue.

Evidence of a possible multicellular life cycle in Escherichia coli

Biofilms are surface-attached multicellular microbial communities. Their genetics have been extensively studied, but the cell-scale morphogenetic events of their formation are largely unknown. Here, we recorded the entirety of morphogenesis in Escherichia coli, and discovered a previously unknown multicellular self-assembly process. Unattached, single-cells formed 4-cell rosettes which grew intoconstant-width chains. After ∼10 cell generations, these multicellular chains attached to surfaces and stopped growing. Chains remained clonal throughout morphogenesis. We showed that this process generates biofilms, which we found are composed of attached clonal chains, aligned in parallel. We investigated genetics of chain morphogenesis: Ag43 facilitates rosette formation and clonality; type-1 fimbriae and curli promote stability and configuration; and extracellular polysaccharide production facilitates attachment. Our study establishes that E.coli, a unicellular organism, can follow a multistage, clonal, genetically-regulated, rosette-initiated multicellular life cycle. These findings have implications for synthetic biology, multicellular development, and the treatment and prevention of bacterial diseases.

Donor-strand exchange drives assembly of the TasA scaffold in Bacillus subtilis biofilms

Many bacteria in nature exist in multicellular communities termed biofilms, where cells are embedded in an extracellular matrix that provides rigidity to the biofilm and protects cells from chemical and mechanical stresses. In the Gram-positive model bacterium Bacillus subtilis, TasA is the major protein component of the biofilm matrix, where it has been reported to form functional amyloid fibres contributing to biofilm structure and stability. Here, we present electron cryomicroscopy structures of TasA fibres, which show that, rather than forming amyloid fibrils, TasA monomers assemble into fibres through donor-strand exchange, with each subunit donating a β-strand to complete the fold of the next subunit along the fibre. Combining electron cryotomography, atomic force microscopy, and mutational studies, we show how TasA fibres congregate in three dimensions to form abundant fibre bundles that are essential for B. subtilis biofilm formation. Our study explains the previously observed biochemical properties of TasA and shows how a bacterial extracellular globular protein can assemble from monomers into β-sheet-rich fibres, and how such fibres assemble into bundles in biofilms.

Abstract C012: Dissecting the reorganization of pancreatic tumor microenvironments after radiation and immunotherapy reveals insights into immunotherapy resistance

Abstract Immune checkpoint blockade (ICB) has revolutionized the treatment of many cancers but has been ineffective for the treatment of microsatellite stable (MSS) PDAC. The lack of efficacy of immunotherapies in PDAC is due to: 1) a desmoplastic tumor microenvironment (TME); 2) the presence of suppressive cells, including myeloid derived suppressor cells and regulatory T cells; and 3) the lack of antigen-presenting dendritic cells (DCs) that are important in priming an effective immune response to generate functionally effective tumor antigen-specific T cells. We recently completed a pilot study of dual ICB (Ipilumamab and Nivolumab) with radiation therapy (SBRT 8Gy for 3 fractions) in a cohort of 25 metastatic PDAC patients that had progressed on conventional chemotherapy; this combination conferred an impressive 18% ORR and 29% disease control rate measured on non-irradiated lesions (historical 0% ORR with ICB in PDAC). This led to a phase 2 study in 30 metastatic PDAC patients using this dual modality treatment paradigm. To understand the role of radiation and ICB in altering the PDAC tumor microenvironment we performed single-cell RNA-sequencing and TCR-sequencing (>180k cells), and single-nucleus RNA-sequencing (>300k cells) on 36 tumor biopsies (23 pre-treatment, 13 paired on-treatment between day 10 and 21) from patients undergoing treatment in our phase 2 study. Tumor tissue was taken from distinct tissue sites, including primary tumors in the pancreas, and liver and abdominal wall metastases. We identified distinct tumor cell state distributions within different tissues, and a redistribution of cells from basal/mesenchymal states to classical states after radiation. We identified several state-specific interferon stimulated gene programs thus cataloging distinct responses of epithelial cells with different transcriptional states. Importantly, we found a redistribution of T cells states towards proliferating and exhausted T cells with unique clonality after radiation. Additionally, the myeloid compartment after radiation was enriched for C1QC+ and MHCII+ macrophage subsets, as well as infiltrating CD16/CD16 monocytes and CD14 monocytes, each showing induction of unique sets of interferon stimulated genes (ISGs). We next sought to better understand immunotherapy resistance mechanisms within these PDAC patients despite finding strong ISG induction in several subsets. We analyzed covarying gene programs and identified multicellular communities of cells before and after radiation that underlie interaction networks associated with radiation. Together our data provides the most comprehensive single-cell atlas of paired biopsies to study tumor and immune cell states in the context of radiation and ICB response. Citation Format: Arnav Mehta, Aparna Parikh, Milan Parikh, Ryan Park, Moshe Sade-Feldman, Lynn Bi, Nicole Carzo, Tarin M. Grillo, Islam Baiev, Olanike Asupoto, Irena Gushterova, Tom LaSalle, Anna Gonye, Emily Blaum, Sebastien Vigneau, Ronan Chaligne, Ana Lako, Thomas Lila, David Nelson, Caroline Porter, Orr Ashenberg, Karthik Jagadesh, William L. Hwang, Christopher Smillie, David P. Ryan, David T. Ting, Theodore Hong, Dana Pe'er, Nir Hacohen. Dissecting the reorganization of pancreatic tumor microenvironments after radiation and immunotherapy reveals insights into immunotherapy resistance [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C012.

Flow cytometric evaluation of yeast-bacterial cell-cell interactions.

Synthetic cell-cell interaction systems can be useful for understanding multicellular communities or for screening binding molecules. We adapt a previously characterized set of synthetic cognate nanobody-antigen pairs to a yeast-bacteria coincubation format and use flow cytometry to evaluate cell-cell interactions mediated by binding between surface-displayed molecules. We further use fluorescence-activated cell sortingto enrich a specific yeast-displayed nanobody within a mixed yeast-display population. Finally, we demonstrate that this system supports the characterization of a therapeutically relevant nanobody-antigen interaction: a previously discovered nanobody that binds to the intimin protein expressed on the surface of enterohemorrhagic Escherichiacoli. Overall, our findings indicate that the yeast-bacteria format supports efficient evaluation of ligand-target interactions. With further development, this format may facilitate systematic characterization and high-throughput discovery of bacterial surface-binding molecules.

Subcellular localization of type IV pili regulates bacterial multicellular development

In mammals, subcellular protein localization of factors like planar cell polarity proteins is a key driver of the multicellular organization of tissues. Bacteria also form organized multicellular communities, but these patterns are largely thought to emerge from regulation of whole-cell processes like growth, motility, cell shape, and differentiation. Here we show that a unique intracellular patterning of appendages known as type IV pili (T4P) can drive multicellular development of complex bacterial communities. Specifically, dynamic T4P appendages localize in a line along the long axis of the cell in the bacterium Acinetobacter baylyi. This long-axis localization is regulated by a functionally divergent chemosensory Pil-Chp system, and an atypical T4P protein homologue (FimV) bridges Pil-Chp signaling and T4P positioning. We further demonstrate through modeling and empirical approaches that subcellular T4P localization controls how individual cells interact with one another, independently of T4P dynamics, with different patterns of localization giving rise to distinct multicellular architectures. Our results reveal how subcellular patterning of single cells regulates the development of multicellular bacterial communities.

Nanoparticle Impact on the Bacterial Adaptation: Focus on Nano-Titania.

Titanium dioxide nanoparticles (nano-titania/TiO2 NPs) are used in different fields and applications. However, the release of TiO2 NPs into the environment has raised concerns about their biosafety and biosecurity. In light of the evidence that TiO2 NPs could be used to counteract antibiotic resistance, they have been investigated for their antibacterial activity. Studies reported so far indicate a good performance of TiO2 NPs against bacteria, alone or in combination with antibiotics. However, bacteria are able to invoke multiple response mechanisms in an attempt to adapt to TiO2 NPs. Bacterial adaption arises from global changes in metabolic pathways via the modulation of regulatory networks and can be related to single-cell or multicellular communities. This review describes how the impact of TiO2 NPs on bacteria leads to several changes in microorganisms, mainly during long-term exposure, that can evolve towards adaptation and/or increased virulence. Strategies employed by bacteria to cope with TiO2 NPs suggest that their use as an antibacterial agent has still to be extensively investigated from the point of view of the risk of adaptation, to prevent the development of resistance. At the same time, possible effects on increased virulence following bacterial target modifications by TiO2 NPs on cells or tissues have to be considered.