The bacterial cytoskeleton modulates motility, type 3 secretion, and colonization in Salmonella.

Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its importance to virulence. In this study we have explored the contribution of the bacterial cytoskeleton to the ability of Salmonella to express and assemble virulence factors and cause disease. The bacterial actin-like protein MreB polymerises into helical filaments and interacts with other cytoskeletal elements including MreC to control cell-shape. As mreB appears to be an essential gene, we have constructed a viable ΔmreC depletion mutant in Salmonella. Using a broad range of independent biochemical, fluorescence and phenotypic screens we provide evidence that the Salmonella pathogenicity island-1 type three secretion system (SPI1-T3SS) and flagella systems are down-regulated in the absence of MreC. In contrast the SPI-2 T3SS appears to remain functional. The phenotypes have been further validated using a chemical genetic approach to disrupt the functionality of MreB. Although the fitness of ΔmreC is reduced in vivo, we observed that this defect does not completely abrogate the ability of Salmonella to cause disease systemically. By forcing on expression of flagella and SPI-1 T3SS in trans with the master regulators FlhDC and HilA, it is clear that the cytoskeleton is dispensable for the assembly of these structures but essential for their expression. As two-component systems are involved in sensing and adapting to environmental and cell surface signals, we have constructed and screened a panel of such mutants and identified the sensor kinase RcsC as a key phenotypic regulator in ΔmreC. Further genetic analysis revealed the importance of the Rcs two-component system in modulating the expression of these virulence factors. Collectively, these results suggest that expression of virulence genes might be directly coordinated with cytoskeletal integrity, and this regulation is mediated by the two-component system sensor kinase RcsC.

Differential Dynamic Microscopy: a High-Throughput Method for Characterizing the Motility of Microorganism

We present a fast, high-throughput method for characterizing the motility of microorganisms in 3D based on standard imaging microscopy. Instead of tracking individual cells, we analyse the spatio-temporal fluctuations of the intensity in the sample from time-lapse images and obtain the intermediate scattering function (ISF) of the system. We demonstrate our method on two different types of microorganisms: bacteria, both smooth swimming (run only) and wild type (run and tumble) Escherichia coli, and the bi-flagellate alga Chlamydomonas reinhardtii. We validate the methodology using computer simulations and particle tracking. From the ISF, we are able to extract (i) for E. coli: the swimming speed distribution, the fraction of motile cells and the diffusivity, and (ii) for C. reinhardtii: the swimming speed distribution, the amplitude and frequency of the oscillatory dynamics. In both cases, the motility parameters are averaged over \approx 10^4 cells and obtained in a few minutes.

Dynamics of filopodium-like protrusion and endothelial cellular motility on onedimensional extracellular matrix fibrils

Endothelial filopodia play key roles in guiding the tubular sprouting during angiogenesis. However, their dynamic morphological characteristics, with the associated implications in cell motility, have been subjected to limited investigations. In this work, the interaction between endothelial cells and extracellular matrix fibrils was recapitulated in vitro, where a specific focus was paid to derive the key morphological parameters to define the dynamics of filopodium-like protrusion during cell motility. Based on one-dimensional gelatin fibrils patterned by near-field electrospinning (NFES), we study the response of endothelial cells (EA.hy926) under normal culture or ROCK inhibition. It is shown that the behaviour of temporal protrusion length versus cell motility can be divided into distinct modes. Persistent migration was found to be one of the modes which permitted cell displacement for over 300 μm at a speed of approximately 1 μm min-1. ROCK inhibition resulted in abnormally long protrusions and diminished the persistent migration, but dramatically increased the speeds of protrusion extension and retraction. Finally, we also report the breakage of protrusion during cell motility, and examine its phenotypic behaviours. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Metabolite and transcriptome analysis of Campylobacter jejuni in vitro growth reveals a stationary-phase physiological switch.

Campylobacter jejuni is a prevalent cause of food-borne diarrhoeal illness in humans. Understanding of the physiological and metabolic capabilities of the organism is limited. We report a detailed analysis of the C. jejuni growth cycle in batch culture. Combined transcriptomic, phenotypic and metabolic analysis demonstrates a highly dynamic 'stationary phase', characterized by a peak in motility, numerous gene expression changes and substrate switching, despite transcript changes that indicate a metabolic downshift upon the onset of stationary phase. Video tracking of bacterial motility identifies peak activity during stationary phase. Amino acid analysis of culture supernatants shows a preferential order of amino acid utilization. Proton NMR (1H-NMR) highlights an acetate switch mechanism whereby bacteria change from acetate excretion to acetate uptake, most probably in response to depletion of other substrates. Acetate production requires pta (Cj0688) and ackA (Cj0689), although the acs homologue (Cj1537c) is not required. Insertion mutants in Cj0688 and Cj0689 maintain viability less well during the stationary and decline phases of the growth cycle than wild-type C. jejuni, suggesting that these genes, and the acetate pathway, are important for survival.

New tools for self-organised pattern formation

Position-dependent gene expression is a critical aspect of the development and behaviour of multicellular organisms. It requires a complex series of interactions to occur between different cell types in addition to intracellular signalling cascades. We used Escherichia coli to study the properties of an artificial signalling system at the interface between two expanding cell populations. We genetically engineered one population to produce a diffusible acyl-homoserine lactone (AHL) signal, and another population to respond to it. Our experiments demonstrate how such a signal can be used to reproducibly generate simple visible patterns with high accuracy in swimming agar. The producing and responding cassettes of two such signalling systems can be linked to produce a symmetric interface for bidirectional communication that can be used to visualise molecular logic. Intracellular feedback between these two cassettes would then create a framework for self-organised patterning of higher complexity. Adapting the experiments of Basu et al. (Basu et al., 2005) using cell motility, rather than a differential response to AHL concentrations as a way to define zones of response, we noted how the interaction of sender and receiver cell populations on a swimming plate could lead to complex pattern formation. Equipping highly motile strains such as E. coli MC1000 with AHL-mediated auto-inducing systems based on Vibrio fischeri luxI/luxR and Pseudomonas aeruginosa lasI/lasR cassettes would allow the amplification of a response to an AHL signal and its propagation. We designed and synthesised codon-optimised auto-inducing luxI/R and lasI/R cassettes as optimal gene expression is crucial for the generation of robust patterns. We still have to complete and test the entire genetic circuitry, although by modelling the system we were able to demonstrate its feasibility. © 2007 The Institution of Engineering and Technology.

Co-ordination of pathogenicity island expression by the BipA GTPase in enteropathogenic Escherichia coli (EPEC).

BipA is a novel member of the ribosome binding GTPase superfamily and is widely distributed in bacteria and plants. We report here that it regulates -multiple cell surface- and virulence-associated -components in the enteropathogenic Escherichia coli (EPEC) strain E2348/69. The regulated components include bacterial flagella, the espC pathogenicity island and a type III secretion system specified by the locus of enterocyte effacement (LEE). BipA positively regulated the espC and LEE gene clusters through transcriptional control of the LEE-encoded regulator, Ler. Additionally, it affected the pattern of proteolysis of intimin, a key LEE-encoded adhesin specified by the LEE. BipA control of the LEE operated independently of the previously characterized regulators Per, integration host factor and H-NS. In contrast, it negatively regulated the flagella-mediated motility of EPEC and in a Ler-independent manner. Our results indicate that the BipA GTPase functions high up in diverse regulatory cascades to co-ordinate the expression of key pathogenicity islands and other virulence-associated factors in E. coli.

BipA: a tyrosine-phosphorylated GTPase that mediates interactions between enteropathogenic Escherichia coli (EPEC) and epithelial cells.

We report the functional characterization of BipA, a GTPase that undergoes tyrosine phosphorylation in an enteropathogenic Escherichia coli (EPEC) strain. BipA mutants adhere to cultured epithelial cells but fail to trigger the characteristic cytoskeletal rearrangements found in cells infected with wild-type EPEC. In contrast, increased expression of BipA enhances actin remodelling and results in the hyperformation of pseudopods. BipA appears to be the first example of a new class of virulence regulator, as it also controls flagella-mediated cell motility and resistance to the antibacterial effects of a human host defence protein. Its striking sequence similarity to ribosome-binding elongation factors suggests that it uses a novel mechanism to modulate gene expression.

Hydrodynamic entrapment of bacteria swimming near a solid surface.

The near-surface motility of bacteria is important in the initial formation of biofilms and in many biomedical applications. The swimming motion of Escherichia coli near a solid surface is investigated both numerically and experimentally. A boundary element method is used to predict the hydrodynamic entrapment of E. coli bacteria, their trajectories, and the minimum separation of the cell from the surface. The numerical results show the existence of a stable swimming distance from the boundary that depends only on the shape of the cell body and the flagellum. The experimental validation of the numerical approach allows one to use the numerical method as a predictive tool to estimate with reasonable accuracy the near-wall motility of swimming bacteria of known geometry. The analysis of the numerical database demonstrated the existence of a correlation between the radius of curvature of the near-wall circular trajectory and the separation gap. Such correlation allows an indirect estimation of either of the two quantities by a direct measure of the other without prior knowledge of the cell geometry. This result may prove extremely important in those biomedical and technical applications in which the near-wall behavior of bacteria is of fundamental importance.

Collective Cell Migration: Leadership, Invasion and Segregation

A number of biological processes, such as embryo development, cancer metastasis or wound healing, rely on cells moving in concert. The mechanisms leading to the emergence of coordinated motion remain however largely unexplored. Although biomolecular signalling is known to be involved in most occurrences of collective migration, the role of physical and mechanical interactions has only been recently investigated. In this paper, a versatile framework for cell motility is implemented in-silico in order to study the minimal requirements for the coordination of a group of epithelial cells. We find that cell motility and cell-cell mechanical interactions are sufficient to generate a broad array of behaviours commonly observed in vitro and in vivo. Cell streaming, sheet migration and susceptibility to leader cells are examples of behaviours spontaneously emerging from these simple assumptions, which might explain why collective effects are so ubiquitous in nature. This analysis provides also new insights into cancer metastasis and cell sorting, suggesting in particular that collective invasion might result from an emerging coordination in a system where single cells are mechanically unable to invade.

Award Winner in the Young Investigator Category, 2014 Society for Biomaterials Annual Meeting and Exposition, Denver, Colorado, April 16-19, 2014: Periodically perforated core-shell collagen biomaterials balance cell infiltration, bioactivity, and mechanical properties.

Orthopedic tissue engineering requires biomaterials with robust mechanics as well as adequate porosity and permeability to support cell motility, proliferation, and new extracellular matrix (ECM) synthesis. While collagen-glycosaminoglycan (CG) scaffolds have been developed for a range of tissue engineering applications, they exhibit poor mechanical properties. Building on previous work in our lab that described composite CG biomaterials containing a porous scaffold core and nonporous CG membrane shell inspired by mechanically efficient core-shell composites in nature, this study explores an approach to improve cellular infiltration and metabolic health within these core-shell composites. We use indentation analyses to demonstrate that CG membranes, while less permeable than porous CG scaffolds, show similar permeability to dense materials such as small intestine submucosa (SIS). We also describe a simple method to fabricate CG membranes with organized arrays of microscale perforations. We demonstrate that perforated membranes support improved tenocyte migration into CG scaffolds, and that migration is enhanced by platelet-derived growth factor BB-mediated chemotaxis. CG core-shell composites fabricated with perforated membranes display scaffold-membrane integration with significantly improved tensile properties compared to scaffolds without membrane shells. Finally, we show that perforated membrane-scaffold composites support sustained tenocyte metabolic activity as well as improved cell infiltration and reduced expression of hypoxia-inducible factor 1α compared to composites with nonperforated membranes. These results will guide the design of improved biomaterials for tendon repair that are mechanically competent while also supporting infiltration of exogenous cells and other extrinsic mediators of wound healing.

How neurons migrate: a dynamic in-silico model of neuronal migration in the developing cortex.

BACKGROUND: Neuronal migration, the process by which neurons migrate from their place of origin to their final position in the brain, is a central process for normal brain development and function. Advances in experimental techniques have revealed much about many of the molecular components involved in this process. Notwithstanding these advances, how the molecular machinery works together to govern the migration process has yet to be fully understood. Here we present a computational model of neuronal migration, in which four key molecular entities, Lis1, DCX, Reelin and GABA, form a molecular program that mediates the migration process. RESULTS: The model simulated the dynamic migration process, consistent with in-vivo observations of morphological, cellular and population-level phenomena. Specifically, the model reproduced migration phases, cellular dynamics and population distributions that concur with experimental observations in normal neuronal development. We tested the model under reduced activity of Lis1 and DCX and found an aberrant development similar to observations in Lis1 and DCX silencing expression experiments. Analysis of the model gave rise to unforeseen insights that could guide future experimental study. Specifically: (1) the model revealed the possibility that under conditions of Lis1 reduced expression, neurons experience an oscillatory neuron-glial association prior to the multipolar stage; and (2) we hypothesized that observed morphology variations in rats and mice may be explained by a single difference in the way that Lis1 and DCX stimulate bipolar motility. From this we make the following predictions: (1) under reduced Lis1 and enhanced DCX expression, we predict a reduced bipolar migration in rats, and (2) under enhanced DCX expression in mice we predict a normal or a higher bipolar migration. CONCLUSIONS: We present here a system-wide computational model of neuronal migration that integrates theory and data within a precise, testable framework. Our model accounts for a range of observable behaviors and affords a computational framework to study aspects of neuronal migration as a complex process that is driven by a relatively simple molecular program. Analysis of the model generated new hypotheses and yet unobserved phenomena that may guide future experimental studies. This paper thus reports a first step toward a comprehensive in-silico model of neuronal migration.