Cell-lysis sensing drives biofilm formation in Vibrio cholerae.

Matrix-encapsulated communities of bacteria, called biofilms, are ubiquitous in the environment and are notoriously difficult to eliminate in clinical and industrial settings. Biofilm formation likely evolved as a mechanism to protect resident cells from environmental challenges, yet how bacteria undergo threat assessment to inform biofilm development remains unclear. Here we find that population-level cell lysis events induce the formation of biofilms by surviving Vibrio cholerae cells. Survivors detect threats by sensing a cellular component released through cell lysis, which we identify as norspermidine. Lysis sensing occurs via the MbaA receptor with genus-level specificity, and responsive biofilm cells are shielded from phage infection and attacks from other bacteria. Thus, our work uncovers a connection between bacterial lysis and biofilm formation that may be broadly conserved among microorganisms.

mSphere of Influence: The complex world of bacterial biogeography.

Drew Bridges works in the field of bacterial signal transduction and studies the formation and disassembly of bacterial biofilms. In this mSphere of Influence article, he reflects on how the paper "Biogeography of a human oral microbiome at the micron scale" by Mark Welch et al. (J. L. Mark Welch, B. J. Rossetti, C. W. Rieken, F. E. Dewhirst, et al., Proc Natl Acad Sci U S A 113:E791-E800, 2016, https://doi.org/10.1073/pnas.1522149113) inspired him to change his research trajectory.

Pneumococcal Extracellular Vesicles Mediate Horizontal Gene Transfer via the Transformation Machinery.

Bacterial cells secrete extracellular vesicles (EVs), the function of which is a matter of intense investigation. Here, we show that the EVs secreted by the human pathogen Streptococcus pneumoniae (pneumococcus) are associated with bacterial DNA on their surface and can deliver this DNA to the transformation machinery of competent cells. These findings suggest that EVs contribute to gene transfer in Gram-positive bacteria, and in doing so, may promote the spread of drug resistance genes in the population.

Synergy between c-di-GMP and Quorum-Sensing Signaling in Vibrio cholerae Biofilm Morphogenesis.

Transitions between individual and communal lifestyles allow bacteria to adapt to changing environments. Bacteria must integrate information encoded in multiple sensory cues to appropriately undertake these transitions. Here, we investigate how two prevalent sensory inputs converge on biofilm morphogenesis: quorum sensing, which endows bacteria with the ability to communicate and coordinate group behaviors, and second messenger c-di-GMP signaling, which allows bacteria to detect and respond to environmental stimuli. We use Vibrio cholerae as our model system, the autoinducer AI-2 to modulate quorum sensing, and the polyamine norspermidine to modulate NspS-MbaA-mediated c-di-GMP production. Individually, AI-2 and norspermidine drive opposing biofilm phenotypes, with AI-2 repressing and norspermidine inducing biofilm formation. Surprisingly, however, when AI-2 and norspermidine are simultaneously detected, they act synergistically to increase biofilm biomass and biofilm cell density. We show that this effect is caused by quorum-sensing-mediated activation of nspS-mbaA expression, which increases the levels of NspS and MbaA, and in turn, c-di-GMP biosynthesis, in response to norspermidine. Increased MbaA-synthesized c-di-GMP activates the VpsR transcription factor, driving elevated expression of genes encoding key biofilm matrix components. Thus, in the context of biofilm morphogenesis in V. cholerae, quorum-sensing regulation of c-di-GMP-metabolizing receptor levels connects changes in cell population density to detection of environmental stimuli. IMPORTANCE The development of multicellular communities, known as biofilms, facilitates beneficial functions of gut microbiome bacteria and makes bacterial pathogens recalcitrant to treatment. Understanding how bacteria regulate the biofilm life cycle is fundamental to biofilm control in industrial processes and in medicine. Here, we demonstrate how two major sensory inputs-quorum-sensing communication and second messenger c-di-GMP signaling-jointly regulate biofilm morphogenesis in the global pathogen Vibrio cholerae. We characterize the mechanism underlying a surprising synergy between quorum-sensing and c-di-GMP signaling in controlling biofilm development. Thus, the work connects changes in cell population density to detection of environmental stimuli in a pathogen of clinical significance.

Signal Transduction Network Principles Underlying Bacterial Collective Behaviors.

Bacteria orchestrate collective behaviors and accomplish feats that would be unsuccessful if carried out by a lone bacterium. Processes undertaken by groups of bacteria include bioluminescence, biofilm formation, virulence factor production, and release of public goods that are shared by the community. Collective behaviors are controlled by signal transduction networks that integrate sensory information and transduce the information internally. Here, we discuss network features and mechanisms that, even in the face of dramatically changing environments, drive precise execution of bacterial group behaviors. We focus on representative quorum-sensing and second-messenger cyclic dimeric GMP (c-di-GMP) signal relays. We highlight ligand specificity versus sensitivity, how small-molecule ligands drive discrimination of kin versus nonkin, signal integration mechanisms, single-input sensory systems versus coincidence detectors, and tuning of input-output dynamics via feedback regulation. We summarize how different features of signal transduction systems allow groups of bacteria to successfully interpret and collectively react to dynamically changing environments.

Quantitative input-output dynamics of a c-di-GMP signal transduction cascade in Vibrio cholerae.

Bacterial biofilms are multicellular communities that collectively overcome environmental threats and clinical treatments. To regulate the biofilm lifecycle, bacteria commonly transduce sensory information via the second messenger molecule cyclic diguanylate (c-di-GMP). Using experimental and modeling approaches, we quantitatively capture c-di-GMP signal transmission via the bifunctional polyamine receptor NspS-MbaA, from ligand binding to output, in the pathogen Vibrio cholerae. Upon binding of norspermidine or spermidine, NspS-MbaA synthesizes or degrades c-di-GMP, respectively, which, in turn, drives alterations specifically to biofilm gene expression. A long-standing question is how output specificity is achieved via c-di-GMP, a diffusible molecule that regulates dozens of effectors. We show that NspS-MbaA signals locally to specific effectors, sensitizing V. cholerae to polyamines. However, local signaling is not required for specificity, as changes to global cytoplasmic c-di-GMP levels can selectively regulate biofilm genes. This work establishes the input-output dynamics underlying c-di-GMP signaling, which could be useful for developing bacterial manipulation strategies.

Does radiology require informed consent for radiation risk?

Recent trends in medical decision-making have moved from paternalistic doctor-patient relations to shared decision-making. Informed consent is fundamental to this process and to ensuring patients' ongoing trust in the health-care profession. It cannot be assumed that patients consent to the risk associated with medical exposures, unless they have been provided with the information to make that decision. This position is supported by both the legal and ethical framework around Radiation Protection detailed in this commentary.

Inverse regulation of Vibrio cholerae biofilm dispersal by polyamine signals.

The global pathogen Vibrio cholerae undergoes cycles of biofilm formation and dispersal in the environment and the human host. Little is understood about biofilm dispersal. Here, we show that MbaA, a periplasmic polyamine sensor, and PotD1, a polyamine importer, regulate V. cholerae biofilm dispersal. Spermidine, a commonly produced polyamine, drives V. cholerae dispersal, whereas norspermidine, an uncommon polyamine produced by vibrios, inhibits dispersal. Spermidine and norspermidine differ by one methylene group. Both polyamines control dispersal via MbaA detection in the periplasm and subsequent signal relay. Our results suggest that dispersal fails in the absence of PotD1 because endogenously produced norspermidine is not reimported, periplasmic norspermidine accumulates, and it stimulates MbaA signaling. These results suggest that V. cholerae uses MbaA to monitor environmental polyamines, blends of which potentially provide information about numbers of 'self' and 'other'. This information is used to dictate whether or not to disperse from biofilms.

Identification of signaling pathways, matrix-digestion enzymes, and motility components controlling Vibrio cholerae biofilm dispersal.

Bacteria alternate between being free-swimming and existing as members of sessile multicellular communities called biofilms. The biofilm lifecycle occurs in three stages: cell attachment, biofilm maturation, and biofilm dispersal. Vibrio cholerae biofilms are hyperinfectious, and biofilm formation and dispersal are considered central to disease transmission. While biofilm formation is well studied, almost nothing is known about biofilm dispersal. Here, we conducted an imaging screen for V. cholerae mutants that fail to disperse, revealing three classes of dispersal components: signal transduction proteins, matrix-degradation enzymes, and motility factors. Signaling proteins dominated the screen and among them, we focused on an uncharacterized two-component sensory system that we term DbfS/DbfR for dispersal of biofilm sensor/regulator. Phospho-DbfR represses biofilm dispersal. DbfS dephosphorylates and thereby inactivates DbfR, which permits dispersal. Matrix degradation requires two enzymes: LapG, which cleaves adhesins, and RbmB, which digests matrix polysaccharides. Reorientation in swimming direction, mediated by CheY3, is necessary for cells to escape from the porous biofilm matrix. We suggest that these components act sequentially: signaling launches dispersal by terminating matrix production and triggering matrix digestion, and subsequent cell motility permits escape from biofilms. This study lays the groundwork for interventions aimed at modulating V. cholerae biofilm dispersal to ameliorate disease.

Cell position fates and collective fountain flow in bacterial biofilms revealed by light-sheet microscopy.

Bacterial biofilms represent a basic form of multicellular organization that confers survival advantages to constituent cells. The sequential stages of cell ordering during biofilm development have been studied in the pathogen and model biofilm-former Vibrio cholerae It is unknown how spatial trajectories of individual cells and the collective motions of many cells drive biofilm expansion. We developed dual-view light-sheet microscopy to investigate the dynamics of biofilm development from a founder cell to a mature three-dimensional community. Tracking of individual cells revealed two distinct fates: one set of biofilm cells expanded ballistically outward, while the other became trapped at the substrate. A collective fountain-like flow transported cells to the biofilm front, bypassing members trapped at the substrate and facilitating lateral biofilm expansion. This collective flow pattern was quantitatively captured by a continuum model of biofilm growth against substrate friction. Coordinated cell movement required the matrix protein RbmA, without which cells expanded erratically. Thus, tracking cell lineages and trajectories in space and time revealed how multicellular structures form from a single founder cell.

Separating Functions of the Phage-Encoded Quorum-Sensing-Activated Antirepressor Qtip.

Quorum sensing is a process of chemical communication that bacteria use to track cell density and coordinate gene expression across a population. Bacteria-infecting viruses, called phages, can encode quorum-sensing components that enable them to integrate host cell density information into the lysis-lysogeny decision. Vibriophage VP882 is one such phage, and activation of its quorum-sensing pathway leads to the production of an antirepressor called Qtip. Qtip interferes with the prophage repressor (cIVP882), leading to host-cell lysis. Here, we show that Qtip interacts with the N terminus of cIVP882, inhibiting both cIVP882 DNA binding and cIVP882 autoproteolysis. Qtip also sequesters cIVP882, localizing it to the poles. Qtip can localize to the poles independently of cIVP882. Alanine-scanning mutagenesis of Qtip shows that its localization and interference with cIVP882 activities are separable. Comparison of Qtip to a canonical phage antirepressor reveals that despite both proteins interacting with their partner repressors, only Qtip drives polar localization.

The intragenus and interspecies quorum-sensing autoinducers exert distinct control over Vibrio cholerae biofilm formation and dispersal.

Vibrio cholerae possesses multiple quorum-sensing (QS) systems that control virulence and biofilm formation among other traits. At low cell densities, when QS autoinducers are absent, V. cholerae forms biofilms. At high cell densities, when autoinducers have accumulated, biofilm formation is repressed, and dispersal occurs. Here, we focus on the roles of two well-characterized QS autoinducers that function in parallel. One autoinducer, called cholerae autoinducer-1 (CAI-1), is used to measure Vibrio abundance, and the other autoinducer, called autoinducer-2 (AI-2), is widely produced by different bacterial species and presumed to enable V. cholerae to assess the total bacterial cell density of the vicinal community. The two V. cholerae autoinducers funnel information into a shared signal relay pathway. This feature of the QS system architecture has made it difficult to understand how specific information can be extracted from each autoinducer, how the autoinducers might drive distinct output behaviors, and, in turn, how the bacteria use QS to distinguish kin from nonkin in bacterial communities. We develop a live-cell biofilm formation and dispersal assay that allows examination of the individual and combined roles of the two autoinducers in controlling V. cholerae behavior. We show that the QS system works as a coincidence detector in which both autoinducers must be present simultaneously for repression of biofilm formation to occur. Within that context, the CAI-1 QS pathway is activated when only a few V. cholerae cells are present, whereas the AI-2 pathway is activated only at much higher cell density. The consequence of this asymmetry is that exogenous sources of AI-2, but not CAI-1, contribute to satisfying the coincidence detector to repress biofilm formation and promote dispersal. We propose that V. cholerae uses CAI-1 to verify that some of its kin are present before committing to the high-cell-density QS mode, but it is, in fact, the broadly made autoinducer AI-2 that sets the pace of the V. cholerae QS program. This first report of unique roles for the different V. cholerae autoinducers suggests that detection of kin fosters a distinct outcome from detection of nonkin.

Micron-scale plasma membrane curvature is recognized by the septin cytoskeleton.

Cells change shape in response to diverse environmental and developmental conditions, creating topologies with micron-scale features. Although individual proteins can sense nanometer-scale membrane curvature, it is unclear if a cell could also use nanometer-scale components to sense micron-scale contours, such as the cytokinetic furrow and base of neuronal branches. Septins are filament-forming proteins that serve as signaling platforms and are frequently associated with areas of the plasma membrane where there is micron-scale curvature, including the cytokinetic furrow and the base of cell protrusions. We report here that fungal and human septins are able to distinguish between different degrees of micron-scale curvature in cells. By preparing supported lipid bilayers on beads of different curvature, we reconstitute and measure the intrinsic septin curvature preference. We conclude that micron-scale curvature recognition is a fundamental property of the septin cytoskeleton that provides the cell with a mechanism to know its local shape.

Experiences of using a commercial dose management system (GE DoseWatch) for CT examinations.

OBJECTIVE: To assess the use of a commercial dose management system (GE DoseWatch; GEMS, Milwaukee, WI) for CT dose management for six common CT examinations. METHODS: Data were acquired over several months using GE DoseWatch for six common CT examinations on three CT scanners. The dose length product (DLP) was taken as the dose indicator. The data were analysed using four different filtering methods: study description, the National Interim Clinical Imaging Procedure code, protocol name and a more detailed filtering method (the reference data set). The filtering methods were compared using an analysis of variance and multiple comparison technique. The different scanners were compared using the reference data set. RESULTS: It was found that integrating DoseWatch with the radiology information system provided improved results compared with using the study description. Filtering by study description was found to be a poor indicator of the mean dose for all three scanners and consistently overestimated (p < 0.05) the head and thorax-abdo-pelvis mean DLP values, despite the large sample sizes. Filtering by the National Interim Clinical Imaging Procedure code or protocol name produced mean DLPs which were not statistically different from the reference data. The scanner intercomparison showed some significant differences between the scanners, usually due to different tube current modulation settings. CONCLUSION: The use of a commercial dose monitoring system provided fast and efficient filtering of substantial amounts of data. The filtering method affected the mean DLP value despite large sample sizes. ADVANCES IN KNOWLEDGE: Dose management systems are relatively new in the UK, and this article shares knowledge on the use of one system.

RNA Controls PolyQ Protein Phase Transitions.

Compartmentalization in cells is central to the spatial and temporal control of biochemistry. In addition to membrane-bound organelles, membrane-less compartments form partitions in cells. Increasing evidence suggests that these compartments assemble through liquid-liquid phase separation. However, the spatiotemporal control of their assembly, and how they maintain distinct functional and physical identities, is poorly understood. We have previously shown an RNA-binding protein with a polyQ-expansion called Whi3 is essential for the spatial patterning of cyclin and formin transcripts in cytosol. Here, we show that specific mRNAs that are known physiological targets of Whi3 drive phase separation. mRNA can alter the viscosity of droplets, their propensity to fuse, and the exchange rates of components with bulk solution. Different mRNAs impart distinct biophysical properties of droplets, indicating mRNA can bring individuality to assemblies. Our findings suggest that mRNAs can encode not only genetic information but also the biophysical properties of phase-separated compartments.

Absolute Arrangement of Subunits in Cytoskeletal Septin Filaments in Cells Measured by Fluorescence Microscopy.

We resolved the organization of subunits in cytoskeletal polymers in cells by light microscopy. Septin GTPases form linear complexes of about 32 nm length that polymerize into filaments. We visualized both termini of septin complexes by single molecule microscopy in vitro. Complexes appeared as 32 nm spaced localization pairs, and filaments appeared as stretches of equidistant localizations. Cellular septins were resolved as localization pairs and thin stretches of equidistant localizations.

Septin Form and Function at the Cell Cortex.

Septins are GTP-binding proteins that form filaments and higher-order structures on the cell cortex of eukaryotic cells and associate with actin and microtubule cytoskeletal networks. When assembled, septins coordinate cell division and contribute to cell polarity maintenance and membrane remodeling. These functions manifest themselves via scaffolding of cytosolic proteins and cytoskeletal networks to specific locations on membranes and by forming diffusional barriers that restrict lateral diffusion of proteins embedded in membranes. Notably, many neurodegenerative diseases and cancers have been characterized as having misregulated septins, suggesting that their functions are relevant to diverse diseases. Despite the importance of septins, little is known about what features of the plasma membrane influence septin recruitment and alternatively, how septins influence plasma membrane properties. Septins have been localized to the cell cortex at the base of cilia, the mother-bud neck of yeast, and branch points of filamentous fungi and dendritic spines, in cleavage furrows, and in retracting membrane protrusions in mammalian cells. These sites all possess some degree of curvature and are likely composed of distinct lipid pools. Depending on the context, septins may act alone or in concert with other cytoskeletal elements to influence and sense membrane properties. The degree to which septins react to and/or induce changes in shape and lipid composition are discussed here. As septins are an essential player in basic biology and disease, understanding the interplay between septins and the plasma membrane is critical and may yield new and unexpected functions.

Quality control within the multicentre perfusion CT study of primary colorectal cancer (PROSPeCT): results of an iodine density phantom study.

OBJECTIVES: To assess the cross-centre consistency of iodine enhancement, contrast-to-noise ratio and radiation dose in a multicentre perfusion CT trial of colorectal cancer. MATERIALS AND METHODS: A cylindrical water phantom containing different iodine inserts was examined on seven CT models in 13 hospitals. The relationship between CT number (Hounsfield units, HU) and iodine concentration (milligrams per millilitre) was established and contrast-to-noise ratios (CNRs) calculated. Radiation doses (CTDIvol, DLP) were compared across all sites. RESULTS: There was a linear relationship between CT number and iodine density. Iodine enhancement varied by a factor of at most 1.10, and image noise by at most 1.5 across the study sites. At an iodine concentration of 1 mg ml(-1) and 100 kV, CNRs ranged from 3.6 to 4.8 in the 220-mm phantom and from 1.4 to 1.9 in the 300-mm phantom. Doses varied by a factor of at most 2.4, but remained within study dose constraints. Iterative reconstruction algorithms did not alter iodine enhancement but resulted in reduced image noise by a factor of at most 2.2, allowing a potential dose decrease of at most 80% compared to filtered back projection (FBP). CONCLUSIONS: Quality control of CT performance across centres indicates that CNR values remain relatively consistent across all sites, giving acceptable image quality within the agreed dose constraints. KEY POINTS: Quality control is essential in a multicentre setting to enable CT quantification. CNRs in a body-sized phantom had the recommended value of at least 1.5. CTDIs and DLPs varied by factors of 1.8 and 2.4 respectively.

Fungal pathogens are platforms for discovering novel and conserved septin properties.

Septins are filament-forming GTP-binding proteins that act as scaffolds in diverse cell functions including division, polarity and membrane remodeling. In a variety of fungal pathogens, it has been observed that septins are required for virulence because cells are unable to survive or are misshapen when septins are mutated. Cell morphology is interconnected with pathogenesis and thus septin mutants displaying aberrant cell morphologies are commonly deficient in host tissue invasion. The degree to which septins orchestrate versus maintain changes in fungal cell morphology during pathogenesis remains to be determined. Aside from the importance of septins in the process of pathogenesis, animal and plant fungal pathogens display complexity in septin form, dynamics, and function not seen in Saccharomyces cerevisiae making these organisms important models for uncovering diversity in septin behavior. Additionally, host septins have recently been implicated in the process of Candida albicans invasion, motivating the need to examine host septins in fungal pathogenesis. Understanding the role of septins in the host-pathogen interaction not only illuminates pathogenesis mechanisms but importantly also expands our understanding of septin biology in general.