Yes / Aeromonas hydrophila is a well-known waterborne pathogen that recently was found to infect humans. Here, we report the draft genome of a freshwater isolate from a Malaysian waterfall, A. hydrophila strain M023, which portrays N-acylhomoserine lactone-dependent quorum sensing. / University of Malaya via High Impact Research Grants (UM C/625/1/HIR/MOHE/CHAN/01, A-000001-50001, and UM C/625/1/HIR/MOHE/CHAN/14/1, H-50001-A000027)

In organisms from insects to vertebrates, Toll-like receptors (TLRs) are primary pathogen detectors that activate downstream pathways, specifically those that direct expression of innate immune effector genes. TLRs also have roles in development in many species. The sea anemone Nematostella vectensis is a useful cnidarian model to study the origins of TLR signaling because its genome encodes a single TLR and homologs of many downstream signaling components, including the NF-κB pathway. In this dissertation, the single N. vectensis TLR (Nv-TLR) is characterized. It is demonstrated that Nv-TLR can activate canonical NF-κB signaling in human cells, the intracellular TIR domain of Nv-TLR can interact with human TLR adapter proteins MAL and MYD88, and the TIR domain of Nv-TLR is required for NF-κB activation. It is shown that the coral pathogen Vibrio coralliilyticus causes a rapidly lethal disease in N. vectensis and that heat-inactivated V. coralliilyticus and bacterial flagellin can activate a reconstituted Nv-TLR-to-NF-κB pathway in human cells. By immunostaining of anemones, Nv-TLR is shown to be expressed in a subset of cnidocytes and many of these Nv-TLR-positive cells also express Nv-NF-κB. Additionally, the nematosome, which is a Nematostella-specific multicellular structure, expresses Nv-TLR, many innate immune pathway homologs, and can engulf V. coralliilyticus. Morpholino knockdown indicates that Nv-TLR also has an essential role during early embryonic development. The characterization of this primitive TLR and identification of a bacterial pathogen for N. vectensis reveal ancient TLR functions and provide a novel model for studying the molecular basis of cnidarian disease and immunity.

Yes / Buruli ulcer (BU) is an insidious neglected tropical disease. Cases are reported around the world but the rural regions of West and Central Africa are most affected. How BU is transmitted and spreads has remained a mystery, even though the causative agent, Mycobacterium ulcerans, has been known for more than 70 years. Here, using the tools of population genomics, we reconstruct the evolutionaryhistoryofM. ulceransbycomparing165isolatesspanning48yearsandrepresenting11endemiccountriesacrossAfrica. The genetic diversity of African M. ulcerans was found to be restricted due to the bacterium’s slow substitution rate coupled with its relatively recent origin. We identified two specific M. ulcerans lineages within the African continent, and inferred that M. ulcerans lineage Mu_A1 existed in Africa for several hundreds of years, unlike lineage Mu_A2, which was introduced much more recently, approximately during the 19th century. Additionally, we observed that specific M. ulcerans epidemic Mu_A1 clones were introduced during the same time period in the three hydrological basins that were well covered in our panel. The estimated time span of the introduction events coincides with the Neo-imperialism period, during which time the European colonial powers divided the African continent among themselves. Using this temporal association, and in the absence of a known BU reservoir or—vector on the continent, we postulate that the so-called "Scramble for Africa" played a significant role in the spread of the disease across the continent. / K.V. was supported by a PhD-grant of the Flemish Interuniversity Council—University Development Cooperation (Belgium). B.d.J. and C.M. were supported by the European Research Council-INTERRUPTB starting grant (no. 311725). T.P.S. was supported by a fellowship from the National Health and Medical Research Council of Australia (1105525). Funding for this work was provided by the Department of Economy, Science and Innovation of the Flemish Government, the Stop Buruli Consortium supported by the UBS Optimus Foundation, and the Fund for Scientific Research Flanders (Belgium) (FWO grant no. G.0321.07N). The computational resources used in this work were provided by the HPC core facility CalcUA and VSC (Flemish Supercomputer Center), funded by the University of Antwerp, the Hercules Foundation and the Flemish Government—department EWI. Aspects of the research in Cameroon and Benin were funded by the Raoul Follereau Fondation France.

Emerging infectious diseases of wildlife have threatened host populations of diverse taxa in recent history, which is largely attributable to anthropogenic global change. In three data chapters, this dissertation examines the drivers of individual- to population-level variation in how host populations respond to novel and emerging pathogens. Each chapter explores these processes in bat populations of North America, predominantly the Northeast and Midwest regions of the United States, impacted by the emerging fungal pathogen that causes white-nose syndrome, Pseudogymnoascus destructans. In Chapter 2, I disentangle the effects of adaptive host traits and environmental influences in driving host population stabilization of the little brown bat (Myotis lucifugus), finding that host-pathogen coexistence in this system is the product of their complex interaction. In Chapter 3, I characterize the range-wide variation in white-nose syndrome impacts on a federally endangered and poorly studied species, the Indiana bat (Myotis sodalis), as well as environmental and demographic determinants of its declines over epidemic time. In Chapter 4, I explore the role of individual variation in roosting microclimate selection of little brown bats in driving their infection severity, yielding important insights into the pathophysiology and environmental dependence of white-nose syndrome. Ultimately, this dissertation characterizes complex drivers of variation in host responses to emerging and invading pathogens, yielding insights essential to the successful mitigation of their impacts. / Doctor of Philosophy / In the same way that Covid-19 swept through our global human population in the year 2020, novel infectious diseases have threatened wildlife populations, sometimes to the point of extinction. Often, however, the processes driving the impacts of novel infectious diseases in wildlife are unknown, despite being important information to protect susceptible populations. In this dissertation, I explore how North American bat populations have been impacted by a recently emerged disease, white-nose syndrome, and what processes cause variation in how individual bats and bat colonies have responded to the disease. In Chapter 2, I explore how the little brown bat (Myotis lucifugus) has evolved to co-exist with its new pathogen and how this coexistence is affected by environmental conditions like temperature and humidity. In Chapter 3, I characterize variation in how populations of the Indiana bat (Myotis sodalis) have responded to white-nose syndrome and how environmental and demographic conditions have affected declines since the disease first emerged. In Chapter 4, I explore how the temperatures used by little brown bats during hibernation affect the severity of their infection, giving us important information on how bats survive with white-nose syndrome and the role of temperature. Altogether, the research in this dissertation describes complex interactions between hosts, pathogens, and their environment in driving the patterns we observe after the emergence of novel infectious diseases.

Au cours de ma thèse je me suis intéressée aux relations hôte-pathogène entre Drosophila melanogaster et Pseudomonas aeruginosa PA14. RhlR, un facteur de transcription bactérien permet à la bactérie d’échapper à la phagocytose. Mon projet de thèse consistait à identifier comment RhlR exerce cette fonction. Mes résultats suggèrent que RhlR exercerait également une fonction indépendante du quorum sensing. Un crible de mutants PA14 nous a permis d’isoler trois gènes importants pour la virulence de la bactérie et possiblement reliés à RhlR: xcpR, vfR et sltB1. L’utilisation de mutants de drosophile tep4, m’a permis de montrer que le rôle d’échappement à la phagocytose se ferait au niveau de la détection de la bactérie. Par ailleurs, mes résultats suggèrent aussi l’intervention d’un composé volatil qui permettrait de synchroniser la virulence de la bactérie. Dans une dernière partie, j’ai étudié les effets d’une co-infection entre un virus entérique et PA14. / During my PhD, I studied the host-pathogen interactions between Drosophila melanogaster and Pseudomonas aeruginosa PA14. We previously identified RhlR as a bacterial transcription factor that allows the bacteria to circumvent phagocytosis. My main PhD project was to study and identify how RhlR exerts this function. My first results suggested that RhlR plays also a role independently its the quorum sensing. A screen of PA14 mutants allowed me to identify three genes involved in PA14 virulence and possibility in RhlR function: xcpR, vfR and sltB1. By using tep4 fly mutants, I have shown that RhlR’s role against phagocytosis is most likely required at the level of PA14 detection. Beside this, my results indicated that possibly a volatile compound is involved to synchronize PA14 virulence. In the last part, I studied the effects of a co-infection between an enteric virus and PA14.

Today billions of people live without access to basic sanitation facilities, and thousands die every week due to diseases caused by fecal contamination associated with improper sanitation. It has thus become crucial for decision makers to have access to relevant and sufficient data to implement appropriate solutions to these problems. The Global Water Pathogen Project http://www.waterpathogens.org/ is dedicated to providing an up-to-date source of data on pathogen reduction associated with different sanitation technologies that are important if the world is to achieve the Sustainable Development Goals (SDGs) related to health and sanitation provision. In this research, a subset of the Global Water Pathogen Project (GWPP) data is used to access the reduction of bacteria and viruses across different mechanical and natural sanitation technologies. The order of expected removal for bacteria during wastewater treatment was reported as highest for a membrane bioreactor (4.4 log10), waste stabilization pond (2.3 log10), conventional activated sludge (1.43 log10), anaerobic anoxic oxic activated sludge (1.9 log10), trickling filter (1.16 log10), and upflow anaerobic sludge blanket reactor (1.2 log10). Furthermore, the order of expected removal for viruses was reported as highest for a membrane bioreactor (3.3 log10), conventional activated sludge (1.84 log10), anaerobic anoxic oxic activated sludge (1.67 log10), waste stabilization pond (1 log10), upflow anaerobic sludge blanket reactor (0.3 log10) and trickling filter (0.29 log10). It was found that hydraulic retention time (HRT) had a statistically significant relation to the reduction of bacteria in an anaerobic, anoxic oxic treatment system. Similarly, a significant relation was found between the number of waste stabilization ponds in series and the expected reduction of bacteria. HRT was also found to be a significant factor in virus reduction in waste stabilization ponds. Additionally, it was observed that waste stabilization ponds, trickling filters, and UASB reactors could obtain a greater reduction in bacteria (5-7 log10) when combined with additional treatment (e.g., chemical disinfection or use of maturation ponds). Also, mechanized systems, such as activated sludge systems and membrane bioreactors, obtained a greater reduction (2-3 log10) of viruses when compared to a natural system. It was concluded that the selection of the best suitable technology for pathogen reduction depends on environmental, design, and operational factors as well as considering the performance of specific wastewater treatment systems individually as well as when combined with other treatment technologies that may provide added removal of microbial constituents.

Dothistromin (DOTH) is a secondary metabolite produced by the fungal peanut pathogen Passalora arachidicola and pine needle pathogen Dothistroma septosporum. The chemical structure of DOTH is similar to a precursor of aflatoxin (AF) and sterigmatocystin (ST), which are secondary metabolites produced by Aspergillus species. A size fractionated genomic library was made and 11 putative DOTH genes were identified in P. arachidicola. The DOTH genes in P. arachidicola were compared to DOTH genes in D. septosporum as well as to AF and ST genes in Aspergillus species. The DOTH gene products in P. arachidicola showed 73 - 96% amino acid identity to DOTH genes in D. septosporum and 50 - 69% amino acid identity to AF/ST genes in Aspergillus. The DOTH biosynthesis genes in P. arachidicola had similar gene organization and direction of transcription to DOTH biosynthesis genes in D. septosporum and is similar in that 11 putative DOTH genes are separated into three mini-clusters. This differs from the AF/ST clusters in which 25 AF/ST genes are tightly clustered in a 70 kb region. Identification of transcription factor binding sites upstream of DOTH genes in P. arachidicola and D. septosporum suggested similar co-regulation of DOTH gene expression in P. arachidicola and D. septosporum. Tandem and inverted repeat sequences were identified in intergenic regions in the P. arachidicola DOTH gene cluster, but the distribution of those repeats appears to be random. This suggests that the fragmentation of the DOTH biosynthesis gene cluster is not due to retrotransposon activity or recombination between repeat sequences. The DOTH biosynthesis gene clusters in P. arachidicola and D. septosporum could be ancestral to AF/ST biosynthesis clusters in Aspergillus species.

<p>Background. Platelets (thrombocytes) are the smallest cells in the blood. Platelet fulfils functions as formation of blood clots when bleeding. Low levels leads to bleeding while high levels increase the risk of thrombosis (obstruction of the circulatory flow system). Platelet transfusions may be required for patients with systemic bleeding and for patients at higher risk of bleeding because of coagulation defects, sepsis (presence of bacteria in the bloodstream), or platelet dysfunction related to medication or disease. A pathogen-reduction system for platelet components would be a useful method since it reduces the risk of bacterial, protozoa, viral and white blood cell contamination. The Intercept Blood System method (IBS) for platelets, destroys DNA and RNA and was validated against the routine method in order to reduce pathogen transmission risk during transfusion. The validation of IBS, the trombocyte count for100 buffy coat concentrates from 2007 were compared to values for 100 buffy coat concentrates from 2006 that had been treated with gamma-radiation. Akademiska sjukhuset in Uppsala has a requirement that 75% of the platelet concentrates contain at least 300*10 9 platelets per unit. IBS fulfilled to 94% compared to 98% for the routine method.</p><p>Thus, the IBS-method was well above the required value and is now used at</p><p>Akademiska sjukhuset in Uppsala.</p>

Background. Platelets (thrombocytes) are the smallest cells in the blood. Platelet fulfils functions as formation of blood clots when bleeding. Low levels leads to bleeding while high levels increase the risk of thrombosis (obstruction of the circulatory flow system). Platelet transfusions may be required for patients with systemic bleeding and for patients at higher risk of bleeding because of coagulation defects, sepsis (presence of bacteria in the bloodstream), or platelet dysfunction related to medication or disease. A pathogen-reduction system for platelet components would be a useful method since it reduces the risk of bacterial, protozoa, viral and white blood cell contamination. The Intercept Blood System method (IBS) for platelets, destroys DNA and RNA and was validated against the routine method in order to reduce pathogen transmission risk during transfusion. The validation of IBS, the trombocyte count for100 buffy coat concentrates from 2007 were compared to values for 100 buffy coat concentrates from 2006 that had been treated with gamma-radiation. Akademiska sjukhuset in Uppsala has a requirement that 75% of the platelet concentrates contain at least 300*10 9 platelets per unit. IBS fulfilled to 94% compared to 98% for the routine method. Thus, the IBS-method was well above the required value and is now used at Akademiska sjukhuset in Uppsala.

The sun is the primary source of all chemical energy on the planet. Starch granules have evolved as storage deposits for captured light energy. Many complex biological functions take place at the starch granule surface, including starch granule metabolism and defense. The starch granule-associated protein puroindoline is a known antimicrobial with unique functional and biological properties, attributed to the presence of a unique tryptophan-rich domain. To test puroindoline's tight association, puroindoline removed from the starch granule surface during water-washing was assessed. Washing more than eight times failed to further reduce puroindoline content of starch granules, suggesting a strong association of puroindoline with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, we used a combination of in situ tryptic digestion and mass spectrometry. We identified the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat. This is the first instance of the tryptophan-rich domain directly observed at the starch granule surface. In addition, using mass spectrometry, we determined that during development and maturation, wheat seeds appear to have resisted infection and lysed the pathogens where, upon desiccation, the molecular evidence remained fixed at the starch granule surface. Proteins with known antimicrobial activity were identified, as well as several proteins from the plant pathogens Agrobacterium tumefaciens, Pectobacterium carotovorum, Fusarium graminearum, Magnaporthe grisea, Xanthomonas axonopodis, and X. oryzae. Future characterization may reveal previously unknown host-pathogen interactions. Finally, we have demonstrated that puroindoline, when expressed in the seeds of transgenic corn, will localize and associate with the starch granule surface in a pattern similar to the puroindoline expression pattern observed in wheat. Surprisingly, puroindoline expression in transgenic corn is correlated with an increase in total seed oil content.