Biofilm formation and toxin production provide a fitness advantage in mixed colonies of environmental yeast isolates

Microbes can engage in social interactions ranging from cooperation to warfare. Biofilms are structured, cooperative microbial communities. Like all cooperative communities, they are susceptible to invasion by selfish individuals who benefit without contributing. However, biofilms are pervasive and ancient, representing the first fossilized life. One hypothesis for the stability of biofilms is spatial structure: Segregated patches of related cooperative cells are able to outcompete unrelated cells. These dynamics have been explored computationally and in bacteria; however, their relevance to eukaryotic microbes remains an open question. The complexity of eukaryotic cell signaling and communication suggests the possibility of different social dynamics. Using the tractable model yeast, Saccharomyces cerevisiae, which can form biofilms, we investigate the interactions of environmental isolates with different social phenotypes. We find that biofilm strains spatially exclude nonbiofilm strains and that biofilm spatial structure confers a consistent and robust fitness advantage in direct competition. Furthermore, biofilms may protect against killer toxin, a warfare phenotype. During biofilm formation, cells are susceptible to toxin from nearby competitors; however, increased spatial use may provide an escape from toxin producers. Our results suggest that yeast biofilms represent a competitive strategy and that principles elucidated for the evolution and stability of bacterial biofilms may apply to more complex eukaryotes

Diffusion-Limited Growth of Microbial Colonies.

The emergence of diffusion-limited growth (DLG) within a microbial colony on a solid substrate is studied using a combination of mathematical modelling and experiments. Using an agent-based model of the interaction between microbial cells and a diffusing nutrient, it is shown that growth directed towards a nutrient source may be used as an indicator that DLG is influencing the colony morphology. A continuous reaction-diffusion model for microbial growth is employed to identify the parameter regime in which DLG is expected to arise. Comparisons between the model and experimental data are used to argue that the bacterium Bacillus subtilis can undergo DLG, while the yeast Saccharomyces cerevisiae cannot, and thus the non-uniform growth exhibited by this yeast must be caused by the pseudohyphal growth mode rather than limited nutrient availability. Experiments testing directly for DLG features in yeast colonies are used to confirm this hypothesis

The role of non-genetic variability in Acute Myeloid Leukaemia

Acute myeloid leukaemia (AML) is a heterogeneous clonal disorder of haematopoietic progenitor cells with a dismal survival. It has a strong reliance on epigenetic and transcriptional factors for disease progression. Accordingly, my lab has previously identified KAT2A, a histone acetyl-transferase, as a requirement for AML maintenance, where chemical inhibition of KAT2A promotes differentiation of AML cell lines (Tzelepis et al., 2016). More recently, using a conditional knockout mouse model for Kat2a our lab showed that it sustains KMT2A/MLLT3 AML stem cells. Kat2a is a classical regulator of transcriptional variability, its loss leading to cell-to-cell heterogeneity in transcription levels, including from genes involved in ribosomal biogenesis and translation (Domingues et al., 2020). No recurrent mutations in the KAT2A gene have been described in AML, and it is unclear if and how it participates in pre-leukaemia-to-AML progression. In this thesis, I studied Kat2a loss in 2 mouse models of AML representing forms of human disease with a prolonged pre-leukaemia phase which typically require additional mutations for leukaemia progression. Specifically, I analysed the biology of RUNX1RUNX1T1(9a) and Idh1R132H-initiated AML in a conditional Kat2aKO background and observed consistent acceleration of leukaemia initiation and progression with perpetuation of transformed Kat2aKO cells in vivo. Single-cell RNA sequencing (scRNA-seq) of early-stage Kat2aWT and Kat2aKO RUNX1-RUNX1T1(9a) pre-leukaemia, suggested an increase in transcriptional variability upon Kat2a loss, which was accompanied by diversification of cell fates towards B-lymphocytes and monocytes. Furthermore, pseudo-temporal ordering of single Kat2aKO cells revealed a highly branched trajectory populated with intermediate stages of transformation, including accumulation of leukaemia progenitors with RUNX1-RUNX1T1 signature. In contrast, Kat2aWT cells displayed a linear haematopoiesis trajectory with minimal branching, and an abrupt transition towards the candidate leukaemia progenitor state. Pathway analysis combined with functional studies indicate a mechanistic contribution of cytoplasmic translation and ribosomal biogenesis-associated genes towards leukaemia progression in both models of pre-leukaemia. Taken together, my work suggests that loss of Kat2a results in accelerated pre-leukaemia transformation accompanied with diversification of cell fate transitions including with increased accessibility to cell states prone to transformation. Furthermore, transformation-prone cells may benefit from low biosynthetic activity to progress to a leukaemic state. I hypothesize that Kat2a loss may function similarly in the context of other malignancies. In the future, this knowledge may aid in the development of early diagnostic tools and suggest bespoke therapeutic interventions

QUANTITATIVE ANALYSIS AND IMAGING-BASED INSIGHTS INTO THE CHARACTERISTICS AND MECHANISMS OF YEAST PATTERN FORMATION

Biofilm formation is a common lifestyle adapted by bacteria and fungi in response to various environmental stresses. Bacterial and fungal biofilms adhering to medical devices convey resistance to antibiotics or biocides, causing high rates of clinical infections. Microorganisms are protected from harsh environmental conditions by reduced stress penetration through the complex biofilm architecture with distinct patterns. Although the molecular regulations of surface patterning have been well characterized in bacteria, the mechanisms underlying the complex pattern formation in eukaryotic biofilms remain unclear. This dissertation aims to investigate the salient features of robust colony expansion in yeast biofilms and the processes driving the complex pattern development. Various salient features of Saccharomyces cerevisiae colony expansion, such as of the change of size, shape, and surface pattern properties were analyzed quantitatively for various combinations of agar and sugar concentrations. I found that the size and irregularity of the FLO11 expressing colony, and wavelength of the pattern were all monotonically decreasing with agar density. These trends were consistent regardless of sugar sources. Using a mathematical model, I also demonstrated that the differential expansion pattern between the center and the edge of the colony due to the spatial differences in glucose concentration affected the convexity of the expansion curve. I found that pattern formation in S. cerevisiae was not caused by localized cell death as in Bacillus subtilis biofilms. Using quantitative measurement and physical models, I found that the surface pattern of S. cerevisiae was consistent with hierarchical wrinkling, determined by the physicochemical properties and thickness of the layered structures of the yeast biofilm and the viscoelastic agar. Furthermore, I found that two-dimensional expansion conferred a competitive advantage for FLO11 sectors during head-to-head competition with flo11Δ cells. Overall these results suggested that two-dimensionality of expansion conveyed by FLO11 directs rapid colony expansion with high irregularity at the rim, hierarchical wrinkling pattern, and competitive advantage during head-to-head competition

Characterizing the shape patterns of dimorphic yeast pseudohyphae

© 2018 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.Pseudohyphal growth of the dimorphic yeast Saccharomyces cerevisiae is analysed using two-dimensional top-down binary images. The colony morphology is characterized using clustered shape primitives (CSPs), which are learned automatically from the data and thus do not require a list of predefined features or a priori knowledge of the shape. The power of CSPs is demonstrated through the classification of pseudohyphal yeast colonies known to produce different morphologies. The classifier categorizes the yeast colonies considered with an accuracy of 0.969 and standard deviation 0.041, demonstrating that CSPs capture differences in morphology, while CSPs are found to provide greater discriminatory power than spatial indices previously used to quantify pseudohyphal growth. The analysis demonstrates that CSPs provide a promising avenue for analysing morphology in high-throughput assays

Intra-colony channel morphology in Escherichia coli biofilms is governed by nutrient availability and substrate stiffness

Nutrient-transporting channels are found throughout mature Escherichia coli biofilms, however the influence of environmental conditions on intra-colony channel formation is poorly understood. We report the effect of different substrate nutrient concentrations and agar stiffness on the structure and distribution of intra-colony channels in mature E. coli colony biofilms using fluorescence mesoscopy and quantitative image analysis. Intra-colony channel width was observed to increase non-linearly with radial distance from the centre of the biofilm and channels were, on average, 50% wider at the centre of carbon-limited biofilms compared to nitrogen-limited biofilms. Channel density also differed in colonies grown on rich and minimal medium substrates, with the former creating a network of tightly packed channels and the latter leading to well-separated, wider channels with easily identifiable edges. We conclude that intra-colony channel morphology in E. coli biofilms is influenced by both substrate composition and nutrient availability

Loss of TET2 in human hematopoietic stem cells alters the development and function of neutrophils

Somatic mutations commonly occur in hematopoietic stem cells (HSCs). Some mutant clones outgrow through clonal hematopoiesis (CH) and produce mutated immune progenies shaping host immunity. Individuals with CH are asymptomatic but have an increased risk of developing leukemia, cardiovascular and pulmonary inflammatory diseases, and severe infections. Using genetic engineering of human HSCs (hHSCs) and transplantation in immunodeficient mice, we describe how a commonly mutated gene in CH, TET2, affects human neutrophil development and function. TET2 loss in hHSCs produce a distinct neutrophil heterogeneity in bone marrow and peripheral tissues by increasing the repopulating capacity of neutrophil progenitors and giving rise to low-granule neutrophils. Human neutrophils that inherited TET2 mutations mount exacerbated inflammatory responses and have more condensed chromatin, which correlates with compact neutrophil extracellular trap (NET) production. We expose here physiological abnormalities that may inform future strategies to detect TET2-CH and prevent NET-mediated pathologies associated with CH

Cooperation and Competition Shape Ecological Resistance During Periodic Spatial Disturbance of Engineered Bacteria

Cooperation is fundamental to the survival of many bacterial species. Previous studies have shown that spatial structure can both promote and suppress cooperation. Most environments where bacteria are found are periodically disturbed, which can affect the spatial structure of the population. Despite the important role that spatial disturbances play in maintaining ecological relationships, it remains unclear as to how periodic spatial disturbances affect bacteria dependent on cooperation for survival. Here, we use bacteria engineered with a strong Allee effect to investigate how the frequency of periodic spatial disturbances affects cooperation. We show that at intermediate frequencies of spatial disturbance, the ability of the bacterial population to cooperate is perturbed. A mathematical model demonstrates that periodic spatial disturbance leads to a tradeoff between accessing an autoinducer and accessing nutrients, which determines the ability of the bacteria to cooperate. Based on this relationship, we alter the ability of the bacteria to access an autoinducer. We show that increased access to an autoinducer can enhance cooperation, but can also reduce ecological resistance, defined as the ability of a population to resist changes due to disturbance. Our results may have implications in maintaining stability of microbial communities and in the treatment of infectious diseases

Rapid and high-resolution analysis of winemaking yeasts using MALDI-TOF MS : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Winemaking is a biologically diverse and dynamic process in which the grape sugar is converted into ethanol, CO2 and other aromatic compounds by yeasts. Saccharomyces cerevisiae is the main species used for wine production, whereas the contribution of non-Saccharomyces yeasts to the distinctiveness of wine was not acknowledged until the 1980s. The indigenous yeasts present in the vineyard mainly belong to non-Saccharomyces species, which can have an important impact on the final wine quality, especially where spontaneous fermentation practices are used. However, metabolic profiles of individual strains of both non-Saccharomyces and Saccharomyces species may differ significantly, and thus lead to different organoleptic properties that are important to increase the expression of terroir in the wine. In this sense, some of these yeast strains may be desirable to be isolated and used for further development of novel wine products. It is also important to identify spoilage yeasts that may contaminate wine with off-flavours. Both cases require the ability to identify yeast strains that contribute particular flavour profiles to the wine. Recently, an emerging proteomic approach of matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been successfully applied to identify yeast species relevant to winemaking. This technology has shown potential in the prediction of the utility of individual yeast strains in the production of different wine styles. Despite this interest, most work focuses on its capacity for clinical identification purposes, and the list of winemaking yeasts in current MALDI-TOF databases is not exhaustive. Furthermore, the predictive potential of this approach has not gone unchallenged. With this in mind, this study aims to further develop MALDI-TOF MS as a rapid and low-cost method for yeast identification and characterisation, as well as assess it as a tool to predict the suitability of individual yeast strains in the production of different wine styles. Based on 14 type strains and 19 field isolates representing 21 yeast species, the efficiency of MALDI-TOF MS for wine yeasts identification was improved by comparing the dried-droplet (DM) and pre-mixing (PM) methods, as well as two mass ranges of m/z 2,000-20,000 and 500-4,000. With this improved protocol, MALDI-TOF MS was used to identify the yeast isolates recovered from the production of Pinot Noir wines that were spontaneously fermented in vineyard versus in winery by an organic wine producer in Waipara, New Zealand. The corresponding MALDI profiles were integrated into our in-house database stored in Software BioNumerics v 7.6. Meanwhile, 26S rRNA sequencing was used in conjunction with Restriction Fragment Length Polymorphism (RFLP) to cross-check the yeast identification results. Afterwards, eight Saccharomyces strains of diverse origin were examined to investigate the influence of growth conditions on MALDI-TOF spectra and to determine the best medium for the use of MALDI-TOF MS to predict wine yeast utility for different wine styles production, including the Pinot Noir grape juice, Chardonnay grape juice, synthetic grape juice, and laboratory-grade artificial culture media (YPD broth and agar). With the pre-selected culture media, YPD agar and YPD broth, a panel of 59 commercial yeasts including 47 wine yeasts and 12 brewing yeasts were then used to validate the predictive potential of MALDI-TOF profiling for individual yeast strains application. Dimensionality reduction techniques (DRTs) of PCA, MDS and UMAP were performed to analyse the data by using BioNumerics v 7.6 and the conda-forge packages for Python. Compared to the routine DM method, PM improved the performance of MALDI-TOF MS on wine-associated yeast analysis and yielded well-defined identification results. This is the first known usage of low-mass range m/z 500-4,000 profiles in winemaking yeast characterisation; this mass range appears unsuitable for the identification at the species level, but may offer some advantages for infraspecific (i.e. strain) classification. This improved MALDI-TOF MS protocol was then successfully applied to indigenous yeast isolated from organically produced Pinot Noir wines for diversity analysis. Thirteen species belonging to eight genera (10 non-Saccharomyces and 3 Saccharomyces yeasts) were identified, with taxonomic diversity reducing as fermentation progressed. MALDI-TOF utility also confirmed the impact of differing production systems on yeast diversity and dynamics of spontaneous fermentation. Furthermore, the MALDI profiles appeared to reflect the impact of different fermentation environments and fermentation stages on individual yeast proteomics. In addition, the yeast cultivation conditions also showed a significant impact on MALDI-TOF profiles, with YPD agar being recommended for taxonomic studies, while YPD broth may offer an improved intra-subspecific differentiation by yielding more discriminatory peaks. MDS and UMAP analyses supported the potential of MALDI-TOF proteomics in predicting the utility of yeast strains in winemaking and brewing sectors, although further studies are necessary to more comprehensively investigate the possible commercial benefits