Insetos são os organismos multicelulares mais bem-sucedidos no ecossistema terrestre e sua riqueza se deve, em parte, à simbiose com microrganismos. Geradora de diversidade fenotípica, a comunidade microbiana associada a insetos permite a manutenção de fenótipos complexos capazes de colonizar novos nichos e de se adaptar a fatores de estresse. A relevância da relação de simbiose na ordem Lepidoptera, todavia, tem sido questionada em função da alta variabilidade da comunidade bacteriana associada aos seus representantes. Tendo como modelo de estudo lagartas do gênero Spodoptera (Lepidoptera: Noctuidae), esse trabalho teve por objetivo melhor compreender a composição e diversidade da microbiota associada ao intestino de lepidópteros, assim como verificar o efeito da pressão de seleção direcionada à resistência na sua estrutura e capacidade de utilização de inseticidas como fonte de carbono. Para isso, foi realizada a análise metagenômica da comunidade microbiana intestinal de quatro espécies do gênero Spodoptera assim como de cinco populações naturais de S. frugiperda e de linhagens suscetível e resistentes desse inseto praga. Foram avaliadas linhagens de S. frugiperda resistentes aos inseticidas spinosad, chlorpyrifos, lambda-cyhalothrin, flubendiamide, teflubenzuron e à toxina de Bacillus thuringiensis Cry1A.105+Cry2Ab2. A análise da composição, riqueza e capacidade da comunidade bacteriana utilizar inseticidas foi baseada na determinação de índices de diversidade alfa e beta, no isolamento de bactérias intestinais em meio seletivo e na análise do potencial de crescimento dos isolados em inúmeros inseticidas. Nossos resultados indicam a importância da microbiota intestinal para esse gênero de Lepidoptera. Bactérias do gênero Enterococcus foram predominantes em todas as populações de Spodoptera analisadas, independentemente da espécie, dieta ou pressões de seleção às quais as lagartas foram expostas. Populações naturais de S. frugiperda apresentaram um microbioma mais diverso e com maior número de bactérias capazes de metabolizar inseticidas. A diversidade do microbioma, assim como a presença e capacidade de bactérias utilizarem inseticidas como fonte de carbono para o seu crescimento foram influenciadas pelo nível de exposição a tais compostos, demonstrando que, assim como o hospedeiro, a microbiota intestinal também se encontra em pressão de seleção direcionada a resistência. / Insects are the most successful multicellular organisms in the terrestrial ecosystem and their richness is partially due to symbiosis with microorganisms. The microbial community associated with insects allows the maintenance of complex phenotypes capable of colonizing new niches and adapting to stressors. The relevance of symbiosis in Lepidoptera, however, has been questioned due to the high variability of the bacterial community associated with its representatives. Using Spodoptera larvae as a model, this work aimed to better understand the composition and diversity of the gut microbiota associated with Lepidoptera, as well as to verify the effects of the selection pressure with insecticides in the microbial gut structure and the ability of members of the community to metabolize insecticides as carbon source. Thus, we performed a metagenomic analysis of the gut microbial community of four species of the genus Spodoptera (Lepidoptera: Noctuidae), as well as for five natural populations of S. frugiperda and susceptible and resistant strains of this pest. Strains of S. frugiperda resistant to the insecticides spinosad, chlorpyrifos, lambda-cyhalothrin, flubendiamide, teflubenzuron and Bacillus thuringiensis toxin Cry1A.105 + Cry2Ab2 were evaluated. The analysis of the composition, richness and ability of the bacterial community to metabolize insecticides were carried out through alpha and beta diversity indexes, isolation of intestinal bacteria in selective medium and analysis of the growth potential of isolates in multiple insecticides. Our data indicate the importance of symbiosis for S. frugiperda. Enterococcus prevailed in all populations analyzed, regardless of the species of Spodoptera, food source or selection pressure that the larvae were exposed. Microbial diversity and ability to metabolize insecticides were higher in natural populations exposed to a range of stressors in the field. The diversity of the gut microbial community associated to Spodoptera frugiperda, as well as the ability of its members to metabolize insecticides, were influenced by the degree of exposure to insecticides, showing that, like the host, the gut microbiota is also under selection pressure to resistance.

One of the main focus of coral reef ecology has been to shed light on the importance of all microbial members of coral holobiont and how their interactions contribute to the coral’s resilience. However, knowledge is lacking about the composition of microbial communities inhabiting the surface mucus layer of corals including Fungia fungites, a species that lives under stressful conditions close to fish farms in Vietnam. I investigated the prokaryotic communities that are thriving in Fungia fungites surface mucus layer (SML) in the wild and how they were affected upon antibiotics and nitrogen stress using 16S rRNA gene-based techniques. Firstly, I observed a significant alteration in the composition of microbial communities due to antibiotics effect, with exposed communities featuring lower richness and α-diversity in contrast to the controls. Further, mucosal microbial communities were found to be mostly dominated by Proteobacteria (especially of the classes of Alphaproteobacteria and Gammaproteobacteria) and less by Bacteroidetes (Flavobacteriia). Results from this study suggest a developed antibiotic resistance of Alteromonadales and Campylobacterales indicated by their increased abundance upon antibiotics effect. Moving forward, future studies should focus on exploring also the contribution of non-prokaryotic microbial members of Fungia fungites holobiont and how antibiotic resistance can potentially influence coral’s health. The results support that Fungia fungites SML microbial communities are strongly affected by antibiotics exposure and call for future research to focus on the function of these microbial communities and how they can contribute to the coral’s resilience.

The endosymbiotic bacteria Wolbachia and the gut microbiome have independently been shown to affect several aspects of insect biology, including reproduction, development, lifespan, stem cell activity and resistance to human pathogens in insect vectors. This research shows that Wolbachia, which reside mainly in the fly germline, affect the microbial species present in the gut of a lab reared strain of Drosophila melanogaster. Fruit flies host two main genera of commensal bacteria – Acetobacter and Lactobacillus. Wolbachia-infected flies have significantly reduced titers of Acetobacter. Analysis of the microbiome of axenic flies fed with equal proportions of both bacteria shows that Wolbachia’s presence is a determinant in the microbiome composition throughout fly development. This effect of Wolbachia on the Drosophila microbiome is host genotype-dependent. To investigate the mechanism of microbiome modulation, the effect of Wolbachia on Imd and ROS pathways, the main regulators of immune response in the fly gut was measured. Wolbachia’s presence did not cause significant gene expression changes of the effector molecules in either pathway. It was also found that Wolbachia slightly reduce the relative length of the acidic region of the gut. However, this observation lacks the robustness necessary to provide a mechanism for the significantly reduced Acetobacter levels. Furthermore, microbiome modulation is not due to direct interaction between Wolbachia and the gut microbes,as confocal microscopy shows that Wolbachia is absent from the gut lumen. These results indicate that the mechanistic basis of the modulation of microbiome composition by Wolbachia is more complex than direct bacteria interaction or Wolbachia’s effect on fly immunity. The findings reported here highlight the importance of considering the gut microbiome composition and host genetic background during Wolbachia-induced phenotypic studies and microbial based-disease vector control strategies.

Although inflammation is a crucial mechanism in response to injury and pathogen clearance, inappropriate or excessive induction of the inflammatory response in pregnancy can cause initiation of the labour cascade and subsequent preterm delivery. Host Defence Peptides (HDPs) have important anti-microbial properties but are also implicated as multifunctional modulators of immunity and infection. They are predominantly secreted by mucosal epithelial cells and released by leukocytes. The specific HDPs that are the focus of this thesis are Human beta-defensin 3 (hBD3) and Human Cathelicidin (hCAP-18/LL-37). The immunomodulatory effect of HDPs in reproductive tissues in response to infection/inflammation has not been well studied. In a pregnant state, the hypothesis of this thesis is that HDPs have a dual role in preventing ascending infection, but also preventing an exacerbated inflammatory response that can cause preterm birth by initiation of the labour cascade. To explore this I determine whether bacterial stimuli can regulate HDPs expression in pregnancy tissues. I also explore what interactions HDPs have on the production/induction of important cytokines that are vital to the inflammatory response. With the aid of HDP knockout mice, the role of these peptides in infection/inflammation and continuation of pregnancy is investigated in a mouse-model of induced preterm-labour. To understand how ascending infection might be controlled by HDPs in pregnancy, I explore how HDPs regulate commensal and pathogenic bacteria. This is achieved by interrogating the maternal microbiome at mucosal sites in HDP knockout animals, utilising the bacterial 16S rRNA gene and next generation sequencing. Results Placental explants respond to Lipopolysaccharide (LPS) challenge by increasing production of pro-inflammatory cytokines. LL-37 but not hBD3 peptide was able to modulate this inflammation by inhibiting the release of these pro-inflammatory cytokines. To establish whether HDPs are critical in the continuation of pregnancy I use a LPS induced mouse–model of preterm labour in animals lacking the genes for the HDPs, Defb14 (Defb14-/-), or Camp (Camp-/-). Intrauterine injection of LPS induced preterm labour in wildtype mice. However, the Defb14-/- and Camp-/- mice do not have an increased rate of preterm labour. Key inflammatory mediators are increased in response to LPS-induced PTL. Camp-/- animals have a similar inflammatory response to wildtype mice when given LPS during pregnancy. To understand how ascending infection might be controlled by HDPs, I interrogated the maternal microbiome at mucosal sites in HDP knockout animals, utilising the bacterial 16S rRNA gene. I established a workflow for 16S rRNA gene sequencing on next-generation sequencing platforms and a bioinformatic pipeline for data analysis. Using this approach I was able to show the mucosal microbiome of Camp-/- animals were significantly different to that of wildtype controls, showing increased diversity in the microbes present. In murine pregnancy, there were very little global cumulative or progressive shifts in bacteria, with the exception of Candidatus arthromitus, which significantly increases with gestation compared to non-pregnancy This thesis has demonstrated that Host Defence Peptides are expressed in pregnancy tissues and have anti-inflammatory properties in response to bacterial stimuli. It is not clear whether the HDPs, hBD3 and LL-37 are fundamental to the immune defence in pregnancy by preventing excessive inflammation, Although, I have shown LL-37 may have a role in modulation of the maternal microbiota.

L’infection broncho-pulmonaire représente le problème majeur des malades atteints de la mucoviscidose. Plusieurs bactéries sont connues depuis des dizaines années comme les principaux agents responsables de ces infections (par exemple Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cepacia, Achromobacter xylosoxidans…). Récemment, certains genres fongiques notamment les champignons filamenteux (comme Aspergillus, Scedosporium…) ont été identifiés comme des pathogènes émergeants ou ré-émergeants pouvant être responsables d’infection invasive. Ainsi, la détection des microorganismes impliqués dans ces colonisations et/ou infections respiratoires demeure importante sur le plan physiopathologique et clinique.Si la culture microbiologique reste la méthode la plus utilisée à ce jour pour le diagnostic des infections microbiennes, elle ne permet pas d’identifier les microbes non-cultivables ou difficiles à cultiver. Depuis quelques années, grâce au développement de la technique moléculaire de séquençage à haut-débit (next generation sequencing ou NGS), plusieurs études ont montré que l’écologie microbienne du poumon des patients atteints de la mucoviscidose est très complexe et correspond à une flore poly-microbienne, appelée le microbiote pulmonaire, comprenant non seulement des bactéries mais également des micromycètes (levures et/ou champignons filamenteux) et des virus et phages. Une dysbiose (modification en abondance et diversité) de cette flore pourrait influencer la fonction respiratoire et l’état clinique du patient.Alors que le microbiome bactérien et son rôle en pathogenèse sont largement étudiés, peu d’études ont porté sur la composante fongique (mycobiote/mycobiome) du microbiote pulmonaire. Notre travail de thèse s’inscrit dans les différents projets développés au sein de l’axe de recherche « Microbiote pro- et eucaryote pulmonaire » coordonné par le Pr Laurence Delhaes dans l’équipe Biologie et Diversité des Pathogènes Eucaryotes Emergeants (BDPEE) dirigée par le Dr Eric Viscogliosi. Il se focalise sur l’analyse NGS du microbiote pro- et eucaryotique respiratoire chez les patients atteints de la mucoviscidose et notamment la comparaison de différentes approches méthodologiques en vue d’une optimisation et standardisation de la méthode.Dans un premier temps, nous présenterons une synthèse des connaissances actuelles d’une part des phénomènes de colonisations/infections fongiques chez les patients atteints de mucoviscidose et d’autre part dans le domaine du microbiote pulmonaire et surtout du mycobiote pulmonaire autour duquel notre équipe se focalise.2Dans un deuxième temps, nous avons travaillé à mieux adapter l’approche NGS aux études du microbiote pulmonaire dans la mucoviscidose. En effet, le séquençage à haut-débit est une technique puissante mais pour laquelle des biais peuvent être introduits à de nombreuses étapes méthodologiques. Un des biais les plus importants est que l’approche NGS ne permet pas de différencier les microorganismes vivants, des cellules mortes ou endommagées, ni de l’ADN extracellulaire. Dans le contexte de notre travail –celui du microbiote pulmonaire chez des patients atteints de mucoviscidose et souvent exposés aux antibiotiques par voie intraveineuse à forte dose, l’analyse NGS pourrait évaluer incorrectement l’abondance et la diversité de ce microbiote pulmonaire. Un prétraitement des échantillons par propidium monoazide (PMA), qui permet de cibler sélectivement l’ADN des cellules vivantes, pourrait être une solution pour palier à cette limite. Notre étude avait donc comme objectif de déterminer si un prétraitement par PMA des expectorations modifiait le microbiote pro- et eucaryote pulmonaire analysé par NGS. Nous discutons l’intérêt et la relevance clinique de cette approche « PMA - NGS » permettant une quantification isolée des microorganismes vivants dans le contexte de la mucoviscidose. / Chronic pulmonary infection results in an irreversible decline in lung function in patients with cystic fibrosis (CF). While several bacteria are known as main causes for these infections (for example: Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cepacia, Achromobacter xylosoxidans...), more recently some fungal genera including filamentous fungi (such as Aspergillus, Scedosporium...) have also been identified as emerging or re-emerging pathogens able to cause invasive mycosis. Thus, the identification of the microorganisms involved in the respiratory colonizations and/or infections has become essential.Still now culture methods remain the gold standard for diagnostic of microbial infections. However, it could not identify non-culturable or difficult-to-cultivate microorganisms. Thanks to the development of high-throughput sequencing (next generation sequencing or NGS), recent studies have shown that the lung of patients with CF is a complex poly-microbial flora, also called the CF lung microbiota, which includes not only bacteria but also fungi (yeast and/or filamentous fungi), and viruses and phages. Dysbiosis (loss of abundance and/or diversity) of the lung microbiota has been associated with the patient's decreased lung function and poor clinical status.While lung bacteriota and its role in pathogenesis have widely been studied, few research studies focus on the fungal component (mycobiota/ mycobiome) of the lungs. Our thesis (PhD work) focuses on NGS analysis of pro- and eukaryotic lung microbiota in CF patients, in particular on the comparison of different methodological approaches to optimize and standardize the NGS protocol. This project has been developed under the supervision of Pr. Laurence Delhaes in the “Biology and Diversity of Eukaryotic Emerging Pathogens” team directed by Dr. Eric Viscogliosi.Firstly, we present a state of art on the current knowledge on the fungal colonization/infections risk in CF as well as the development of new concepts of lung microbiota and lung mycobiota on which our team focuses.Secondly, we applied the NGS approach to study the pro- and eukaryotic microbiota in the sputum samples of CF patient lung. Indeed, NGS is a powerful technique that may introduce biases on numerous methodological steps. One of the most important biases is that this technique could not differentiate among the living microorganisms, the dead or damaged cells, and the extracellular DNA. In the context of the CF lung microbiota which is often exposed to high-dose intravenous antibiotics, the analysis by NGS might evaluate4inaccurately the abundance and the diversity of the lung microbiota. Pretreatment of samples by propidium monoazide (PMA), which can target selectively the DNA of viable cells, could be a solution to overcome this limitation. Our study aimed to determine whether a sample pretreatment with PMA modified the lung pro- and eukaryotic microbiota analyzed by NGS. We discuss the clinical relevance of this approach "PMA - NGS" in the context of CF patients to a better quantification of living microorganisms.

Metagenomics, or genomic sequence of the community of microbiota (bacteria, fungi, virus), enables an investigation of the full complement of genetic material, including virulence, antibiotic resistance, and strain differentiating markers. The granularity to distinguish between closely related strains is important as within one species, these strains possess distinct functions and relationships to a host. To analyze metagenomic samples, I developed a reference-based approach that utilizes both single nucleotide variants and genetic content to assign species and strain-level designations. After refining this approach with complex simulated communities, I utilized it to analyze the microbial communities present in skin samples from healthy and diseased individuals. First, to investigate strain-level heterogeneity in healthy adults, I focused on the common skin commensals Propionibacterium acnes and Staphylococcus epidermidis with well-documented sequence variation. Results indicated that an individual’s strains of P. acnes are shared across multiple sites of his or her body, and that those strains are more similar within than between individuals. For S. epidermidis, in addition to individual site similarities, there were also site-specific strains. Overall these results emphasize that both individuality and site specificity shape our bodies’ microbial communities. Based on longitudinal data, an individual’s strain signatures remain stable for up to a year despite external, environmental perturbations. I then used metagenomic data to explore microbial temporal dynamics in atopic dermatitis (AD; eczema), an inflammatory skin disease commonly associated with Staphylococcal species. Species-level investigation of AD flares demonstrated a microbial dichotomy in which S. aureus predominated on more severely affected patients while S. epidermidis predominated on less severely affected patients. Strain-level analysis determined that S. aureus-predominant patients were monocolonized with distinct S. aureus strains, while all patients had heterogeneous S. epidermidis strain communities. To assess the host immunologic effects of these species, I topically applied patient-derived strains to mice. AD strains of S. aureus were sufficient to elicit a skin immune response, characteristic of AD patients. This suggests a model whereby staphylococcal strains contribute to AD progression through activation of the host immune system. Overall, this strain-level analysis of healthy and disease communities provides previously unexplored resolution of human skin microbiome. / 2018-03-24T00:00:00Z

The gut microbiome is a community of closely interacting microbes living in the gastrointestinal tract. Its structure has direct relevance to health. Disturbances to the microbiome, such as due to antibiotic use, have been implicated in various diseases. The goal of this study was to determine how the gut microbiome reacts to and recovers from disturbance caused by antibiotics. Because diet also influences the microbiome, this study included the interaction between diet and antibiotics. Half of the mice in each diet treatment were given antibiotics to disturb their microbiomes. After cessation of antibiotics, mice were paired in combinations within diets to determine whether the microbiomes of control mice influenced the disturbed microbiomes of formerly antibiotic mice. Chapter 1. Diet significantly altered the structure of the gut microbiome but its effect was significantly smaller than the effect of antibiotics. There was a significant interaction between diet and antibiotics; the antibiotic effect was larger in the cornstarch diet than in the glucose diet. Dysbiotic microbiomes resulting from antibiotics were characterized by an increase in Bacteroidetes and Proteobacteria, and a decrease in Firmicutes. Antibiotic administration also resulted in an initial increase OTU diversity, mainly because it reduced the abundance of dominant OTUs, resulting in greater evenness. Chapter 2. Seven weeks after the cessation of antibiotics (experiment termination), the effect of the antibiotics on the microbiome was still evident. The structure of the dysbiotic microbiome had not returned to that of control mice. Antibiotics significantly increased the relative abundance of some taxa and significant decreased the relative abundance of others. It was unexpected that the taxonomic hierarchy within the microbiome did not recover after 7 weeks following cessation of antibiotics. It would appear, therefore, that antibiotics established a new, semi-stable hierarchy. Chapter 3. When paired together, the assumption was that dysbiotic microbiomes of antibiotic mice would be positively influenced by microbiomes of control mice, based on the assumption that the control mouse would act as a probiotic for the antibiotic mouse, either via coprophagy or consumption of food contaminated by feces. Contrary to that hypothesis, the microbiomes of control mice became more similar to that of antibiotic mice. One can offer at least two hypotheses to explain this result, but neither was tested. First, compared to the control microbiome, the dysbiotic microbiome may have been more stable and thus more resistant to change due to invasion by OTUs from the control microbiome. Other research has shown that dysbiotic microbiomes have a high degree of stability. If this were true, the use of probiotics is questionable. Second, one or more of the antibiotics could still have been active at the initial phase of pairing, and coprophagy caused the microbiome of the control mice to rapidly become dysbiotic. If this is true, the experiment should have been conducted with a waiting period between the cessation of antibiotic administration and pairing.

abstract: The prevalence of obesity and obesity-related disorders have increased world-wide. In the last decade, the intestinal microbiome has become a major indicator of metabolic and gastrointestinal health. Previous research has shown that high-fat diet (HFD) consumption can alter the microbial composition of the gut by increasing the abundance of gram-positive bacteria associated with the onset of obesity and type 2 diabetes. Although, the most common form of obesity and metabolic syndrome intervention is exercise and diet, these recommendations may not improve severe cases of obesity. Thus, an important relevance of my project was to investigate whether the intake of an organometallic complex (OMC) would prevent the onset of metabolic and gastrointestinal complications associated with high-fat diet intake. I hypothesized that the consumption of a HFD for 6 weeks would promote the development of metabolic and gastrointestinal disease risk factors. Next, it was hypothesized that OMC treatment would decrease metabolic risk factors by improving insulin sensitivity and decreasing weight gain. Finally, I hypothesized that HFD-intake would increase the abundance of gram-positive bacteria associated with gastrointestinal disease. My preliminary data investigated the effects of a 6-week HFD on the development of hepatic steatosis, intestinal permeability and inflammation in male Sprague Dawley rats. I found that a 6-week HFD increases hepatic triglyceride concentrations, plasma endotoxins and promotes the production of pro-inflammatory cytokines in the cecum wall. I then investigated whether OMC treatment could prevent metabolic risk factors in male Sprague-Dawley rats fed a HFD for 10 weeks and found that OMC can mitigate risk factors such hyperglycemia, liver disease, impaired endothelial function, and inflammation. Lastly, I investigated the effects of a 10-week HFD on the gastrointestinal system and found an increase in liver triglycerides and free glycerol and alterations of the distal gut microbiome. My results support the hypothesis that a HFD can promote metabolic risk factors, alter the gut microbiome and increase systemic inflammation and that OMC treatment may help mitigate some of these effects. Together, these studies are among the first to demonstrate the effects of a soil-derived compound on metabolic complications. Additionally, these conclusions also provide an essential basis for future gastrointestinal and microbiome studies of OMC treatment. / Dissertation/Thesis / Doctoral Dissertation Biology 2019

The atypical antipsychotic risperidone is associated with weight gain and cardio-metabolic side effects. In light of growing evidence implicating the gut microbiome in the host’s energy regulation and in xenobiotic metabolism, it is hypothesized that risperidone-induced weight gain is mediated through alterations in the gut microbiome. The impact of chronic and short-term risperidone treatment on the gut microbiome of pediatric, psychiatric patients was examined in a cross-sectional and prospective design. Chronic treatment with risperidone was associated with a significant increase in body mass index (BMI) and a significant reduction in the ratio of Bacteroidetes to Firmicutes, as compared to naïve psychiatric controls. Predictive metagenomic analyses, indicate that gut microbiota dominating the risperidone-treated patients are enriched for pathways, such as short-chain fatty acid production, which have been implicated in weight gain. Alterations in the microbiome due to risperidone treatment were further demonstrated in wild-type female mice and shown to be a result of a reduction in host energy expenditure. Risperidone-treated mice exhibit significant weight gain and an altered gut microbiome relative to controls while maintaining normal food intake behavior and digestive efficiency, indicating that increased weight gain is due to reduced energy expenditure. Moreover, fecal transfer from risperidone-treated mice to a second cohort of naïve mice was performed via daily gastric gavage and aerobic and non-aerobic resting metabolic rates (RMR) were monitored using combined calorimetry. This transfer has no effect on aerobic RMR in recipients, but induces a significant suppression of non-aerobic RMR in mice receiving stool from risperidone-treated donors establishing a causal effect of the altered gut microbiome upon energy expenditure. Finally, daily transfer of phage, a subset of the gut microbiome, isolated from the gut of risperidone treated donors was also sufficient to cause excess weight gain in naïve recipients animals through suppression of energy expenditure. Together, these data highlight a major role for the gut microbiome for weight gain following chronic use of risperidone, and demonstrate that the mechanism depends upon suppression of energy expenditure.

The Leishmania spp. are kinetoplastid protozoan parasites that cause a spectrum of highly prevalent and neglected tropical diseases known as leishmaniasis. The parasites must undergo two life forms during their life cycle: the extracellular promastigote life stage within the sand fly vector, and the intracellular amastigote life stage after internalization of host phagocytic cells. In the extracellular life stage, Leishmania promastigotes reside and develop to their infectious metacyclic form solely in the gut lumen of the sand fly, a process known as metacyclogenesis. During this process, other organisms that co-inhabit the sand fly gut, collectively known as the microbiome, influence parasite development. Based on the hypothesis that vector gut microbiota influence the development of parasite virulence, we sequenced midgut microbiomes of the sand fly Lutzomyia longipalpis with or without L. infantum infection. Sucrose fed sand flies contained a highly diverse, stable midgut microbiome. Blood feeding caused a decrease in bacterial richness, which eventually recovered. However, bacterial richness progressively decreased in L. infantum-infected sand flies. Furthermore, parasites altered the relative abundance of several bacterial phylogenies, including Pseudomonas and Serratia. Importantly, antibiotic-mediated perturbation of the midgut microbiome rendered sand flies unable to support parasite growth and consequent development to infectious metacyclic forms, and revealing the level of microbial diversity may induce flies resistant to infection. Together, these data suggest the sand fly midgut microbiome is a critical factor for Leishmania growth and differentiation prior to disease transmission. During the intracellular amastigote life form, macrophages are the primary cell type to phagocytize parasites. The effect of secreted factors such as exosomes from Leishmania-infected human cells and their effect on the immune response has not been extensively investigated. In this thesis, we characterized the proteome of primary human donor monocyte-derived macrophage (MDM) exosomes during L. infantum infection compared to donor-matched uninfected controls, and determined their impact on naïve MDMs measured by cytokine gene expression and resistance to subsequent parasite infection. Proteomic comparisons of infected and uninfected MDM exosomes were made using stable isotopic dimethyl labeling LC-MS/MS technology. A total of 484 human proteins were identified between four donors. Proteins significantly less abundant in exosomes derived from infected MDMs were matrix metalloprotease 9, galectin-3 binding protein, and several Annexins and histone proteins. Proteins more abundant included galectin-1, galectin-9, and serotransferrin and transferrin receptor 1. Interestingly, class I and class II MHC protein chains were differentially abundant in our samples. Furthermore, we observed several Leishmania spp. proteins in exosomes from infected MDMs as well. Naïve MDMs pretreated with exosomes from infected or uninfected MDM for 4 hours were not more resistant to L. infantum infection nor displayed increased gene expression of the pro-inflammatory cytokines IL-1α, IL-1β, IL-6, IL-8 or TNF-α. To date, the work presented in this thesis is the first to comprehensively identify the proteome in primary human MDM exosomes during Leishmania spp. infection, and to determine the impact of these exosomes on the immune response of other naïve human MDMs.