Reconstructing the Forest of Lineage Trees of Diverse Bacterial Communities Using Bio-inspired Image Analysis

Cell segmentation and tracking allow us to extract a plethora of cell attributes from bacterial time-lapse cell movies, thus promoting computational modeling and simulation of biological processes down to the single-cell level. However, to analyze successfully complex cell movies, imaging multiple interacting bacterial clones as they grow and merge to generate overcrowded bacterial communities with thousands of cells in the field of view, segmentation results should be near perfect to warrant good tracking results. We introduce here a fully automated closed-loop bio-inspired computational strategy that exploits prior knowledge about the expected structure of a colony's lineage tree to locate and correct segmentation errors in analyzed movie frames. We show that this correction strategy is effective, resulting in improved cell tracking and consequently trustworthy deep colony lineage trees. Our image analysis approach has the unique capability to keep tracking cells even after clonal subpopulations merge in the movie. This enables the reconstruction of the complete Forest of Lineage Trees (FLT) representation of evolving multi-clonal bacterial communities. Moreover, the percentage of valid cell trajectories extracted from the image analysis almost doubles after segmentation correction. This plethora of trustworthy data extracted from a complex cell movie analysis enables single-cell analytics as a tool for addressing compelling questions for human health, such as understanding the role of single-cell stochasticity in antibiotics resistance without losing site of the inter-cellular interactions and microenvironment effects that may shape it

Άρθρο στα πρακτικά ενός Συνεδρίου Παγκόσμιας εμβέλειας στο οποίο θα παρουσιάζονται τα αποτελέσματα των τεχνικών της ανάλυσης εικόνας (Π24-Δ5.1)

Ανάλυση έκθεση

Segmentation and tracking individual pseudomonas aeruginosa bacteria in dense populations of motile cells

The dynamics of individual bacteria underlies the manifestation of complex multicellular behaviours such as biofilm development and colony expansion. High resolution movies of expanding bacterial colonies reveal intriguing patterns of cell motions. A quantitative understanding of the observed behaviour in relation to the bacteria's own motile apparatus and to hydrodynamic forces requires that bacteria be identified and tracked over time. This represents a demanding undertaking as their size is close to the diffraction limit; they are very close to each other; and a typical image may contain over a thousand cells. Here, we describe the approach that we have developed to segment individual bacteria and track them in high resolution phase contrast microscopy movies. We report that over 99% of non-overlapping bacteria could be segmented correctly using mathematical morphology, and we present preliminary results that exploit this new capability. © 2009 IEEE

Segmentação, crescimento e seguimento de células Escherichia coli em imagens microscópicas

Este trabalho tem como finalidade a obtenção do seguimento celular, sendo importante para os profissionais da área da microbiologia, visto que auxilia na contagem de células e na evolução e crescimento das mesmas de modo a diminuir os erros de contagem e seguimento manual das células. Neste projeto aborda-se o crescimento celular de células Escherichia coli (E.coli), a segmentação de células e o seguimento celular. Primeiramente analisou-se o software schnitzcells que é um programa de seguimento de células e deste modo procedeu-se a um estudo na tentativa de obter melhores resultados. Desta forma, através de modificações no processo de segmentação obtiveram-se melhores resultados do que no programa schnitzcells. De seguida, aplicou-se o método de sobreposição de imagens aos pares de modo que os centróides das células na imagem seguinte estivessem sobrepostos com os centróides das células na imagem anterior e assim proceder-se ao seguimento celular. No seguimento celular verificou-se os centróides dos objetos na imagem seguinte encontravam-se no interior de uma célula na imagem anterior e também calculou-se a distância mínima entre objetos, uma vez que existiam células não sobrepostas e deste modo obteve-se o seguimento celular. Assim, constata-se melhores resultados na metodologia implementada, visto que o processo realiza-se de forma automática enquanto no software schnitzcells necessita de correções manuais a nível da segmentação e do seguimento celular para obterem-se resultados corretos.This work aims to obtain the cell tracking, being important to the professionals in the area of microbiology, as it aids in the counting of cells and in the evolution and growth of them in order to decrease the errors of counting and manual tracking of cells. On this thesis it is described Escherichia Coli (E.coli) cellular growth, cellular segmentation and its tracking. At first it was conducted an analysis of the software schnitzcells, which is a program that tracks cells so that a study in an attempt to achieved better results could be executed. In this way, through some modifications in the process of segmentation, better results were indeed achieved than the ones presented in the program schnitzcells. Afterwards, the method of overlapping images was applied in pairs so that the centroids of the cells in the next image were overlapping with the centroids in the previous image in order to ensue to the tracking of the cells. In the process of tracking the cells, it was verified whether the centroids of the objects in the next image were inside a cell in the previous image. Thus, it was calculated the minimum distance between objects and it was obtained the segmentation of each cell. In short, better results are obtained in the implemented methodology, since the process is performed automatically while with the software schnitzcells manual corrections are required in the segmentation and in the cellular tracking in order to obtain correct results

Αναλυτική έκθεση υπολογιστικών μοντέλων για την προσομοίωση της συμπεριφοράς μικροβιακών κοινοτήτων που σχηματίζουν βιοϋμένια (Π23-Δ5.1 και Δ5.2)

Περιλαμβάνεται ο ακόλουθος πίνακας: 1. Εισαγωγή 2. Υπάρχουσες προσεγγίσεις (State of the art) 2.1. Υπάρχοντα λογισμικά επεξεργασίας κυτταρικών ταινιών 2.2. Μοντελοποίηση 3. Μεθοδολογία Μοντελοποίησης 4. Τεχνική Προσέγγιση 4.1. Ανάλυση κυτταρικών ταινιών 4.2. Μελέτη μεμονωμένων κυττάρων 4.2.1. Βάση δεδομένων για τα κυτταρικά χαρακτηριστικά 4.2.2. Οπτικοποίηση και μελέτη χαρακτηριστικών μεμονωμένων κυττάρων 4.3. Υπολογιστική μοντελοποίηση 4.3.1. Σενάρια μοντελοποίησης 4.3.2. Αποτελέσματα Μοντελοποίησης 5. Σύνοψη Βιβλιογραφί

The spatial and temporal characterisation of functional interactions between the key membrane stress proteins at the single molecule level in live Escherichia coli cells

All the cell types must maintain the integrity of their membranes which is important for cell viability. The membrane structure and function is maintained by many different response mechanisms. One such unique bacterial membrane stress response is the Phage shock protein response (Psp). It is composed of a transcriptional activator, negative regulator, signal sensors and transducers and stress effectors. Using milli second time-scale single-molecule fluorescence microscopy in live E. coli cells, the localisations, two dimensional diffusion dynamics and stoichiometry of functional Psp proteins were determined under non-stress and membrane stress conditions. The two major proteins studied in this research are a bacterial enhancer binding protein phage shock protein F (PspF) and the negative regulator and major effector phage shock protein A (PspA). For the imaging studies stable and functional chromosomal fusions of PspF and PspA to Venus fluorescent protein were used replacing the native proteins. It was established that a stable repressive PspF-PspA complex is located in the nucleoid and PspF displays DNA associated diffusion dynamics similar to other DNA-binding transcription factors such as LacI. PspF as a hexamer activated a single psp promoter at a time. The effector V-PspA assembled as higher order oligomers localised at the lateral membrane and showed very slow dynamics. In addition in vivo and in vitro structural studies of PspA showed that N-terminal amphipathic helix governed the balance between the dual functions of PspA. In a proposed model of the cellular landscape of the Psp response, the PspF-PspA inhibitory complex localised at the nucleoid transiently communicated with the polar regions of cells occupied by PspBC under non-stress conditions. With the stress conditions PspA retained at the polar membrane along with PspBC, while PspF associated with the active transcription complex to initiate the expression of psp genes. With the increase in the amounts of PspA, it organised itself into higher order effector moving along the MreB directed helical path contacting with the membrane via RodZ and in this way facilitated the organisation of membrane repair pathways at the damaged sites.Open Acces

Applications of bacterial enterotoxins, ribosome-inactivating proteins and viral cytotoxins

A toxin can be described as a foreign substance that inflicts damages to living organisms. Naturally occurring proteinaceous toxins can derive from bacteria, fungi, plants, animal venoms and even viruses. Identifying the toxins’ underlying mechanisms of action has been a major research interest in order to develop inhibitors against their effects. Nonetheless, various findings have sparked the use of toxic moieties for the medical benefit resulting in treatment options as for example for cancers. To gain novel insights into the structure and function of a toxin, the toxin itself has to be synthesized. In vivo production can involve high laboratory safety standards as well as a low total protein amount since the toxin might harm the overexpressing cell. An alternative to circumvent these drawbacks is cell-free protein synthesis (CFPS). Within this doctoral thesis CFPS was established as a platform technology for the production and application of proteinaceous toxins in diagnostic and medical fields. As a first step, various bacterial toxins were analyzed. The mechanisms of action of the tripartite pore-forming toxins (PFT) Hbl and Nhe were studied by hemolytic activity assays, cell-based toxicity assessments and electrophysiological recordings. Next, the PFT CytK was analyzed to identify its potential as a biological nanopore that can be used as a diagnostic tool. This thesis identified the CytK1 variant as a candidate for a nanopore development. Further, two AB5 toxins, namely the cholera toxin and the heat-labile enterotoxin, were modified. These modified toxins could be fluorescently labeled and tested for their functional activity. These data are a proof-of-concept for using CPFS for intracellular trafficking of toxins and coupling of payloads for drug delivery. In a second step, a targeted toxin combining the plant-derived toxin Dianthin and the epidermal growth factor (EGF) was assessed for its potency as a potential cancer therapeutic. The medical benefit of this Dianthin-EGF targeted toxin was demonstrated on human squamous cell carcinoma samples. 0.1 nM Dianthin-EGF in combination with an endosomal escape enhancer suppressed the growth of carcinoma colonies by almost 50%. As a third and last step, CFPS was assessed for its potential as a rapid response system against novel viral pathogens using SARS-CoV2 viral proteins. All SARS-CoV2 proteins could be synthesized and analyzed. The cytotoxic behaviors of the nsp1 and envelope protein were determined. The nucleocapsid protein was quantitatively detected by specific antibodies thereby facilitating cell-free systems for the validation of available antibodies. All in all, this thesis successfully developed a platform technology for the cell-free synthesis, functional characterization and application of toxic proteins in clinical and diagnostic fields

Metabolic diversity in cell populations: probability densities over the flux polytope

Even in clonal populations, cells appear to be strongly heterogeneous in terms of, e.g., protein levels, RNA levels, sizes at birth or division, interdivision times and elongation rates. Part of this variability is likely due to the inherent stochasticity of gene expression at the level of single cells. It is however known that heterogeneous populations may possess an evolutionary advantage, for instance in variable environments or under stress. Despite appearing to be at odds with the idea of optimality presented in the previous chapters, metabolic diversity can be described and modeled within the constraint-based framework introduced in the previous chapters. Specifically, a statistical representation of heterogeneous populations can be obtained by defining suitable probability distributions on the flux polytope. This chapter addresses • the different sources of variation that affect microbial metabolism along with the mechanisms that may favor higher variability, • the methods devised to represent heterogeneous microbial populations within the framework of constraint- based models, and • how these approaches connect to the optimality scenario presented in the previous chapters

Structural and functional studies of the terminal protein complex of the human complement system

The membrane attack complex (MAC: C5blC61C71C81C9n) is the terminal complex of the mammalian complement cascade. Its principal function is to form a self-assembling, potentially cytolytic pore spanning the membrane of a foreign cell. However, inappropriate complement activation and subsequent aberrant MAC action is responsible for tissue damage in several human pathologies.1 The C-terminal domains or modules that are exclusive to the C6 and C7 components of the MAC, form a putative molecular arm. These modules consist of two complement control protein modules (CCPs) followed by a pair of Factor I-like Modules (FIMs). The C-terminal molecular arms of C6 and C7 are important for linking the complement activation cascade with MAC assembly and thereby ensuring MAC assembly occurs only when needed. They interact with the C345C domain of C5 and C5b. Currently there are no high-resolution structural data for any of the MAC components but efforts are underway to solve the structures of the individual domains. For instance, the structure of the central MAC/perforin domain of C8 has been determined, so has the structure of C5 and the FIM domains of C7. However , the lack of structural information for the MAC is being balanced by an increased understanding of mechanism, and eventually towards the rational design of therapies designed to supress MAC formation.This report describes the preparation from bacterial and yeast cells of ¹³C,¹⁵N-labelled samples of the C7 CCP-pair and of a triple module consisting of the second CCP followed by the FIM-pair, as well as other constructs from the C6 and C7 C-terminal arms. The NMR samples were used to solve 3D solutions of structures, thus allowing for reconstruction of the four-module C-terminus of C7. Efforts were aimed at obtaining additional structural information for C7. This included SAXS analysis as well as the development of a novel approach using chemical cross-linking followed by tryptic digestion and mass spectrometry-based identification of cross-linked peptides