Toxoplasma gondii ist ein obligat intrazellulärer protozoischer Parasit, der Toxoplasmose beim Menschen und bei Warmblütern verursacht. Die Signalübertragung durch zyklische Nukleotide ist entscheidend für das erfolgreiche intrazelluläre Überleben und die Vermehrung der Parasiten. Hier haben wir die physiologische und biochemische Bedeutung der wesentlichen Phosphodiesterasen (PDEs) in Toxoplasma gondii Tachyzoiten untersucht. Durch C-terminale Markierung von 18 PDEs konnten wir die Expression von 11 PDEs nachweisen. Die Immunogold-Färbung zeigte, dass TgPDE1, TgPDE2 und TgPDE9 im gesamten Parasitenkörper verteilt sind, einschließlich des inneren Membrankomplexes, des apikalen Pols, der Plasmamembran, des Zytosols, der dichten Granula und der Rhoptry, was auf eine räumliche Kontrolle der Signalübertragung innerhalb der Tachyzoiten hindeutet. Anschließend stellten wir fest, dass die meisten Enzyme berüchtigte dual-spezifische Phosphodiesterasen sind, wobei TgPDE2 anders als T.gondii cAMP-spezifisch ist, während T.gondii keine cGMP-spezifische Phosphodiesterase besitzt. Unsere enzymkinetischen Daten zeigen, dass TgPDE2 die höchste Affinität zu seinem Substrat aufweist, während die dualen PDEs (TgPDE1, TgPDE7 und TgPDE9) eine höhere Affinität zu cGMP als zu cAMP haben. Ein Screening der Hemmung gängiger PDE-Inhibitoren auf TgPDEs ergab, dass TgPDE1 das Ziel von BIPPO und Zaprinast ist. Darüber hinaus ergab die biologische Bedeutung, dass TgPDE1 und TgPDE2 einzeln für das Wachstum des Parasiten notwendig sind und ihr Verlust zum Tod des Parasiten führt, was auf ihre funktionelle Redundanz hindeutet. Darüber hinaus identifizierten wir Kinasen und Phosphatasen innerhalb der TgPDE1- und TgPDE2-Interaktome, die die enzymatische Aktivität über Protein-Protein-Interaktionen oder posttranslationale Modifikationen steuern könnten. Insgesamt unterstreichen unsere Erkenntnisse über die subzelluläre Lokalisierung, die katalytische Funktion, die medikamentöse Hemmung und die physiologische Bedeutung der wichtigsten Phosphodiesterasen die unvorhersehbare Plastizität und das therapeutische Potenzial der zyklischen Nukleotid-Signalübertragung in T. gondii. Der Datensatz der cAMP-bindenden Interaktoren, den wir in einem anderen Aspekt dieser Studie offengelegt haben, wird wertvolle Einblicke in die allgegenwärtige Natur der cAMP-vermittelten Signalübertragung in T. gondii Tachyzoiten liefern. / Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis in human and warm-blood organisms. Cyclic nucleotide signaling is crucial for the successful intracellular survival and replication of the parasites. Here, we dissected the physiological and biochemical importance of the essential phosphodiesterases (PDEs) in Toxoplasma gondii tachyzoite. By C-terminal tagging of 18 PDEs, we detected the expression of 11 PDEs. Immunogold staining revealed that TgPDE1, TgPDE2 and TgPDE9 are distributed throughout the parasite body, including the inner membrane complex, the apical pole, the plasma membrane, the cytosol, dense granules, and rhoptry, suggesting the spatial control of signaling within tachyzoites. Subsequently, we identified that most enzymes are notorious dual-specific phosphodiesterases, and TgPDE2 is cAMP specific differently, whilst T.gondii lacks of cGMP specific phosphodiesterase. Our enzyme kinetic data demonstrated that the highest affinity to its substrate belongs to TgPDE2, while the dual PDEs (TgPDE1, TgPDE7 and TgPDE9) have higher affinity with cGMP than cAMP. Inhibition screening of commonly-used PDE inhibitors on TgPDEs, signifying TgPDE1 as the target of BIPPO and zaprinast. Furthermore, the biological significance revealed TgPDE1 and TgPDE2 are individually necessary for parasite growth, and their loss associatively results in parasite death, implying their functional redundancy. In addition, we identified kinases and phosphatases within the TgPDE1 and TgPDE2 interactomes, which may operate the enzymatic activity via protein-protein interactions or posttranslational modifications. Collectively, our findings on subcellular localization, catalytic function, drug inhibition, and physiological relevance of major phosphodiesterases highlight the unforeseeable plasticity and therapeutic potential of cyclic nucleotide signaling in T. gondii. The data set of cAMP-binding interactors, which we disclosed in another aspect of this study, will provide valuable insight into the pervasive nature of cAMP-mediated signaling in T. gondii tachyzoites.

<p>  </p> <p>Epstein-Barr virus (EBV) is a complex oncogenic symbiont. The molecular mechanisms governing EBV carcinogenesis remain elusive and the functional interactions between virus and host cells are incompletely defined. Some of the known mechanisms include viral integration into the host genome, expression and mutation(s) of viral genes and the host response to the virus. Despite decades of research there is a lack of effective treatment options for EBV-positive cancer patients underscoring an urgent need to further investigate the mechanisms underlying tumorigenesis as well as explore and develop personalized treatment strategies for patients with EBV-positive cancers. In Chapter 1, I introduce Epstein-Barr Virus (EBV), the two phases of EBV lifecycle and an overview of certain EBV-associated carcinomas. I will also discuss the underlying mechanisms and few current therapeutic strategies against EBV infection. Next, I will discuss some of the preclinical model systems and high-throughput computation techniques that are commonly used by researchers in the field of EBV.  </p> <p>In chapter 2, we have systematically analyzed RNA-sequencing from >1000 patients with 15 different cancer types, comparing virus and host factors of EBV+ to EBV- tissues to reveal novel insights into EBV-positive tumors. First, we observed that EBV preferentially integrates at highly accessible regions of the cancer genome with significant enrichment in super-enhancer architecture. Second, we determined that the expression of twelve EBV transcripts, including LMP1 and LMP2, correlated inversely with EBV reactivation signature. Over-expression of these genes significantly suppressed viral reactivation, consistent with a ‘Virostatic’ function. Third, we identified hundreds of novel frequent missense and nonsense variations in Virostatic genes in cancer samples, and that the variant genes failed to regulate their viral and cellular targets in cancer. Lastly, we were able to dichotomously classify EBV-positive tumors based on patterns of host interferon signature genes and immune checkpoint markers, such as PD-L1 and IDO1. </p> <p>In chapter 3, we probed the lifecycle of EBV on a cell-by-cell basis using single cell RNA sequencing (scRNA-seq) data from six EBV-immortalized lymphoblastoid cell lines (LCL). While the majority of LCLs comprised cells containing EBV in the latent phase of its life cycle, we identified two additional clusters that had distinct expression of both host and viral genes. Both clusters were high expressors of EBV Latent Membrane Protein-1 (LMP1) but differed in their expression of other EBV lytic genes, including glycoprotein gene GP350. We further probed into the transcriptional landscape of these clusters to identify potential regulators which will be discussed in further detail in the chapter. Importantly, I was able to demonstrate enhancing HIF1-a signaling by using Pevonedistat, a compound that stabilized HIF1-a can preferentially induce the transcriptional program specific to one of the three identified clusters. </p> <p>In Chapter 4, I describe some of my recent work. In this project, we have used an intuitive <em>in-silico </em>drug prediction approach to rapidly screen and identify FDA-approved or clinically available compounds that can be repurposed to induce lytic cycle in different EBV+ tumors. Using this strategy, we identified Ciclopirox, an antifungal drug, as a potent inducer of lytic cycle in EBV+ epithelial cancers. We used EBV+ GC cells to determine the effect of Ciclopirox on EBV reactivation as well as identify the underlying mechanisms. In summary, we discovered that reactivation of EBV lytic cycle by Ciclopirox is mediated by multiple pathways, two of the major ones being the HIF1-a and NF-kB pathways. Although, Ciclopirox treatment enhanced the killing effect of antiviral, further investigation is needed to effectively deliver this drug <em>in vivo.</em> Throughout this chapter, I have discussed findings that needs further investigation and proposed necessary experiments. Finally, in Chapter 5 I have summarized my work and described how our work can provide novel insights that can help delineate some of the complexities of host-pathogen interactions in EBV-associated malignancies. </p>

Quando le condizioni sono sfavorevoli alla crescita, membri dei generi Bacillus e Clostridia (incluso Clostridium botulinum, l’agente eziologico del botulismo) formano endospore, forme cellulari estremamente resistenti, metabolicamente dormienti e difficili da distruggere. Tuttavia, le spore attraverso il processo di germinazione, riattivano il ciclo vegetativo non appena le condizioni tornano favorevoli. Questa capacità di “riattivazione” delle spore è causa di “food spoilage” e di intossicazioni alimentari. Considerando che le specie Clostridium botulinum e Clostridium sporogenes sono filogeneticamente correlate, in questo lavoro, il ceppo Clostridium sporogenes UC9000, isolato da latte crudo, è stato utilizzato come modello non-patogeno di Clostridium botulinum per studiare la germinazione. Studi fisiologici hanno rivelato che le spore del ceppo UC9000 germinano in presenza di L-alanina/ L-cisteina in combinazione con L-lattato, mentre un analisi in silico ha permesso di identificare omologhi dei recettori coinvolti nella risposta all’L-alanina in Bacillus. Attraverso l’analisi del genoma sono stati identificati gli enzimi SleB, CwlJ e SleL, responsabili della degradazione del cortex. CwlJ è stato localizzato nel coat della spora grazie ad uno studio di proteomica, è stato espresso in forma solubile in E. coli ed un test di attività in vitro ha evidenziato la sua capacità di indurre la germinazione di spore “decoated” / When environmental conditions are unfavorable to the growth, Bacillus and Clostridium bacteria (including Clostridium botulinum, the causative agent of foodborne botulism) form endospores, metabolically dormant cell types resistant to several adverse conditions and difficult to kill. However, under suitable conditions, spores resume the vegetative life by triggering the germination process. Thus, spores are dangerous agents of human foodborne disease and food spoilage. In this work, the strain Clostridium sporogenes UC9000, isolated from raw milk, was used like not-pathogenic model of Clostridium botulinum to better understand the mechanisms underpinning the Clostridium germination. Clostridium sporogenes is a species phylogenetically related to Clostridium botulinum and often used like its surrogate. Physiological studies revealed that UC9000 spores germinate in presence of L-alanine/L-cysteine in combination with L-lactate, while in silico analyses allowed the identification of homologues of the Bacillus germinant receptors responsive to L-alanine. The genome screening also detected genes coding for SleB, CwlJ and SleL, enzymes participating to the cortex degradation. CwlJ was found resident in the spore coat by performing a proteomic analysis, it was expressed in soluble form in E. coli and an in vitro assay of activity revealed its capability to induce germination when added exogenously to decoated spores

Le virus Epstein-Barr (VEB) est un pathogène opportuniste qui a la capacité d’immortaliser les lymphocytes B et de provoquer une prolifération maligne, appelée syndrome lymphoprolifératif post-transplantation (SLP), chez les individus immunodéprimés. A l’intérieur de ce groupe, les personnes à plus haut risque sont les enfants, puisqu’ils sont à risque de développer une infection primaire par le VEB pendant leur régime d’immunosuppression post-greffe. Dans le but de développer un anticorps préventif, notre laboratoire s’est attardé au rôle du cycle lytique du VEB dans le développement du SLP. À cette fin, le premier objectif du présent projet vise à fournir la preuve expérimentale de l’existence ou non d’une phase réplicative productive pendant l’infection aiguë des lymphocytes B sanguins. Un examen des événements qui se déroulent au tout début de l’infection par le VEB tant au niveau de la réplication virale qu’au niveau de l’expression des gènes lytiques précoces et tardifs a révélé l’existence d’une phase réplicative productive pendant l’infection aiguë. Ceci a permis de justifier l’élaboration, dans notre laboratoire, d’un anticorps chimère (murin-humain) neutralisant, dirigé contre la protéine gp350 située sur l’enveloppe virale. Le deuxième objectif, quant à lui, vise à fournir la preuve expérimentale de la capacité neutralisante de cet anticorps chimère. Des essais de caractérisation in vitro ont démontré une capacité de reconnaissance de la protéine cible, notamment la gp350, et une capacité de neutralisation du virus par l’anticorps chimère. L’anticorps chimère anti-gp350 pourra faire l’objet d’essais précliniques in vivo en vue d’évaluer sa capacité à reconnaître le virus et à prévenir l’apparition de tumeurs de type SLP chez les souris SCID. Il pourrait être éventuellement utilisé, par la suite, comme traitement préemptif contre les tumeurs dans l’espoir de mieux gérer les patients à risque de développer un SLP. / Epstein-Barr virus (EBV) is an opportunistic pathogen in immunocompromised transplant patients. In these patients EBV infection can lead to malignant B-cell lymphoproliferation, called post-transplant lymphoproliferative disease (PTLD). This thesis project aimed to investigate the role of lytic EBV infection in the genesis of PTLD. The first experimental objective was to provide in vitro proof that EBV could induce productive replication upon acute in vitro infection of B cells. Data obtained through study of viral DNA replication and transcription during the first 96 hours post-infection indicate that lytic infection does occur. These results provided justification for proceeding to the second experimental objective which involved the characterization of an anti-gp350 human-mouse chimeric antibody for its capacity to recognize and neutralize EBV. Results showed that this antibody did possess neutralization activity. Further study of this anti-gp350 chimeric antibody in SCID mice is necessary in order to evaluate its in vivo efficacy against PTLD.

Les oligonucléotides (ONs) antisens présentent un fort potentiel en tant qu’agents thérapeutiques. Toutefois, leurs propriétés physicochimiques limitent leur utilisation en thérapie génique. Pour pallier aux divers obstacles, des systèmes de vectorisation, tels que les micelles polyioniques (PICMs), ont été développés. Grâce à leur structure unique, les micelles protégent l’ON contre une dégradation prématurée et le couplage d’un ligand à leur surface augmente leur spécificité et leur internalisation. Dans d’autres systèmes, un polymère adjuvant aux propriétés pH-sensibles peut être ajouté pour faciliter la sortie de l’endosome et augmenter l’efficacité de l’ON. L’objectif général de ce mémoire était de mettre au point des PICMs ternaires ciblées pour l’administration d’ONs. Ces micelles assureraient à la fois l’internalisation cellulaire de leur cargaison en interagissant avec des récepteurs cellulaires et sa fuite de l’endosome grâce à un mécanisme de déstabilisation de la membrane endosomale. Pour cela, des PICMs composées d’un copolymère cationique de type poly(éthylène glycol)-bloc-poly(méthacrylate d’(alkylamino)éthyle) et d’un copolymère d’acide méthacrylique ont été préparées. Les propriétés physicochimiques de ces vecteurs ont démontré qu’ils permettaient une condensation efficace de l’acide nucléique et ce, indépendamment de la nature du polymère cationique et de l’acide nucléique. Finalement, une approche de couplage par pont disulfure a été développée afin de greffer au copolymère un fragment d’anticorps dirigé contre les récepteurs de la transferrine. En conclusion, ces travaux démontrent la versatilité et le potentiel des PICMs ternaires en tant que vecteurs d’acide nucléique, et proposent une méthodologie de couplage d’un ligand afin de formuler des PICMs ciblées. / Antisens oligonucleotides (ONs) present great potential as therapeutic agents. However, their physicochemical properties hinder their use in gene therapy. Targeting systems, such as polyion complex micelles (PICMs), have been proposed to circumvent the main hurdles related to ON delivery. Their unique core/shell structure can protect the ON against premature degradation and the coupling of a ligand on their surface can increase their specificity and internalization. In other systems, a polymer with pH-sensitive properties can be added to facilitate the release of the ON from the endosome and increase its efficiency. The present work was aimed at optimizing ternary PICMs targeted for the delivery of antisens ON. Such systems would provide both cellular internalization of cargo by interaction with receptors on the surface of cell membranes and escape from the endosome through a mechanism of destabilization of the endosomal membrane. PICMs composed of cationic copolymers of poly(ethylene glycol)-bloc-poly((alkylamino)ethyl methacrylate) with a methacrylic acid copolymer adjuvant were prepared. Their physicochemical properties suggest that efficient complexation of nucleic acids was obtained, regardless of the nature of the cationic polymer and the nature of the nucleic acid. Finally, a synthetic approach was developed for the conjugation of an antibody fragment directed against the transferrin receptor via a labile disulfide bond at the end of the cationic copolymer. In conclusion, the work presented herein displays the versatility and potential of ternary PICMs as vehicles for the delivery of ONs and also provides a method for the conjugation of a ligand to generate targeted ternary PICMs.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Le virus Epstein-Barr (VEB) est un pathogène opportuniste qui a la capacité d’immortaliser les lymphocytes B et de provoquer une prolifération maligne, appelée syndrome lymphoprolifératif post-transplantation (SLP), chez les individus immunodéprimés. A l’intérieur de ce groupe, les personnes à plus haut risque sont les enfants, puisqu’ils sont à risque de développer une infection primaire par le VEB pendant leur régime d’immunosuppression post-greffe. Dans le but de développer un anticorps préventif, notre laboratoire s’est attardé au rôle du cycle lytique du VEB dans le développement du SLP. À cette fin, le premier objectif du présent projet vise à fournir la preuve expérimentale de l’existence ou non d’une phase réplicative productive pendant l’infection aiguë des lymphocytes B sanguins. Un examen des événements qui se déroulent au tout début de l’infection par le VEB tant au niveau de la réplication virale qu’au niveau de l’expression des gènes lytiques précoces et tardifs a révélé l’existence d’une phase réplicative productive pendant l’infection aiguë. Ceci a permis de justifier l’élaboration, dans notre laboratoire, d’un anticorps chimère (murin-humain) neutralisant, dirigé contre la protéine gp350 située sur l’enveloppe virale. Le deuxième objectif, quant à lui, vise à fournir la preuve expérimentale de la capacité neutralisante de cet anticorps chimère. Des essais de caractérisation in vitro ont démontré une capacité de reconnaissance de la protéine cible, notamment la gp350, et une capacité de neutralisation du virus par l’anticorps chimère. L’anticorps chimère anti-gp350 pourra faire l’objet d’essais précliniques in vivo en vue d’évaluer sa capacité à reconnaître le virus et à prévenir l’apparition de tumeurs de type SLP chez les souris SCID. Il pourrait être éventuellement utilisé, par la suite, comme traitement préemptif contre les tumeurs dans l’espoir de mieux gérer les patients à risque de développer un SLP. / Epstein-Barr virus (EBV) is an opportunistic pathogen in immunocompromised transplant patients. In these patients EBV infection can lead to malignant B-cell lymphoproliferation, called post-transplant lymphoproliferative disease (PTLD). This thesis project aimed to investigate the role of lytic EBV infection in the genesis of PTLD. The first experimental objective was to provide in vitro proof that EBV could induce productive replication upon acute in vitro infection of B cells. Data obtained through study of viral DNA replication and transcription during the first 96 hours post-infection indicate that lytic infection does occur. These results provided justification for proceeding to the second experimental objective which involved the characterization of an anti-gp350 human-mouse chimeric antibody for its capacity to recognize and neutralize EBV. Results showed that this antibody did possess neutralization activity. Further study of this anti-gp350 chimeric antibody in SCID mice is necessary in order to evaluate its in vivo efficacy against PTLD.

Les oligonucléotides (ONs) antisens présentent un fort potentiel en tant qu’agents thérapeutiques. Toutefois, leurs propriétés physicochimiques limitent leur utilisation en thérapie génique. Pour pallier aux divers obstacles, des systèmes de vectorisation, tels que les micelles polyioniques (PICMs), ont été développés. Grâce à leur structure unique, les micelles protégent l’ON contre une dégradation prématurée et le couplage d’un ligand à leur surface augmente leur spécificité et leur internalisation. Dans d’autres systèmes, un polymère adjuvant aux propriétés pH-sensibles peut être ajouté pour faciliter la sortie de l’endosome et augmenter l’efficacité de l’ON. L’objectif général de ce mémoire était de mettre au point des PICMs ternaires ciblées pour l’administration d’ONs. Ces micelles assureraient à la fois l’internalisation cellulaire de leur cargaison en interagissant avec des récepteurs cellulaires et sa fuite de l’endosome grâce à un mécanisme de déstabilisation de la membrane endosomale. Pour cela, des PICMs composées d’un copolymère cationique de type poly(éthylène glycol)-bloc-poly(méthacrylate d’(alkylamino)éthyle) et d’un copolymère d’acide méthacrylique ont été préparées. Les propriétés physicochimiques de ces vecteurs ont démontré qu’ils permettaient une condensation efficace de l’acide nucléique et ce, indépendamment de la nature du polymère cationique et de l’acide nucléique. Finalement, une approche de couplage par pont disulfure a été développée afin de greffer au copolymère un fragment d’anticorps dirigé contre les récepteurs de la transferrine. En conclusion, ces travaux démontrent la versatilité et le potentiel des PICMs ternaires en tant que vecteurs d’acide nucléique, et proposent une méthodologie de couplage d’un ligand afin de formuler des PICMs ciblées. / Antisens oligonucleotides (ONs) present great potential as therapeutic agents. However, their physicochemical properties hinder their use in gene therapy. Targeting systems, such as polyion complex micelles (PICMs), have been proposed to circumvent the main hurdles related to ON delivery. Their unique core/shell structure can protect the ON against premature degradation and the coupling of a ligand on their surface can increase their specificity and internalization. In other systems, a polymer with pH-sensitive properties can be added to facilitate the release of the ON from the endosome and increase its efficiency. The present work was aimed at optimizing ternary PICMs targeted for the delivery of antisens ON. Such systems would provide both cellular internalization of cargo by interaction with receptors on the surface of cell membranes and escape from the endosome through a mechanism of destabilization of the endosomal membrane. PICMs composed of cationic copolymers of poly(ethylene glycol)-bloc-poly((alkylamino)ethyl methacrylate) with a methacrylic acid copolymer adjuvant were prepared. Their physicochemical properties suggest that efficient complexation of nucleic acids was obtained, regardless of the nature of the cationic polymer and the nature of the nucleic acid. Finally, a synthetic approach was developed for the conjugation of an antibody fragment directed against the transferrin receptor via a labile disulfide bond at the end of the cationic copolymer. In conclusion, the work presented herein displays the versatility and potential of ternary PICMs as vehicles for the delivery of ONs and also provides a method for the conjugation of a ligand to generate targeted ternary PICMs.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Mounting concerns about drug-resistant pathogenic bacteria have rekindled the interest in bacteriophages (bacterial viruses). As bacteria’s natural predators, bacteriophages offer a critical advantage over antibiotics, namely that they can be highly specific. This means that phage therapeutics can be designed to destroy only the infectious agent(s), without causing any harm to our microbiota. However, the potential secondary effects on the balance of microbiota through bacteriophage-induced genome evolution remains as one of the critical apprehensions regarding phage therapy. There exists a significant gap in knowledge regarding the direct and indirect effect of phage therapeutics on the microbiota. The aim of this thesis was to: (1) establish an in vivo model for investigation of the evolutionary dynamics and co-evolution of therapeutic phage and its corresponding host bacterium in the gut; (2) determine if phage therapy can affect the composition of the gut microbiota, (3) observe the differences of phage-resistant bacteria mutants evolved in vivo in comparison to those evolved in vitro. We used germ-free mice colonized with a consortium of eight known bacteria, known as the altered Schaedler flora (ASF). The colonizing strain of choice (mock infection) was a non-pathogenic strain E. coli K-12 (JM83) known to co-colonize the ASF model, which was challenged in vivo with T7 phage (strictly lytic). We compared the composition of the gut microbiota with that of mice not subject to phage therapy. Furthermore, the resistant mutants evolved in vivo and in vitro were characterized in terms of growth fitness and motility. / Thesis / Master of Applied Science (MASc) / Bacteriophages are viruses that infect bacteria. After their discovery in 1917, bacteriophages were a primary cure against infectious disease for 25 years, before being completely overshadowed by antibiotics. With the rise of antibiotic resistance, bacteriophages are being explored again for their antibacterial activity. One of the critical apprehensions regarding bacteriophage therapy is the possible perturbations to our microbiota. We set out to explore this concern using a simplified microbiome model, namely germ-free mice inoculated with only 8 bacteria plus a mock infection challenged with bacteriophage. We monitored this model for 9 weeks and isolated a collection of phage-resistant bacterial mutants from the mouse gut that developed post phage challenge, maintaining the community of mock infection inside the gut. A single dose of lytic phage challenge effectively decreased the mock infection without causing any extreme long-term perturbations to the gut microbiota.