No description available.

Chronic inflammation is associated with the pathophysiology of obstetrical disorders (e.g. preterm birth [PTB]) and gynecological diseases (e.g. endometriosis); however, the exact mechanism(s) for these conditions are unknown. Numerous immunological conditions and disease states (e.g. inflammatory bowel disease, Crohn’s disease, systemic lupus erythematomus) also disrupt the microbiome homeostasis by inducing a number of changes in the microbial flora when compared to that of healthy individuals. Furthermore, the gastrointestinal (GI) microbiome is one of the principal regulators of circulating estrogens which are known to directly impact the female reproductive disorders endometriosis and PTB. Thus, an alteration of microbial species could indicate a shift in immune balance from homeostatic to pro-inflammatory, and an aberrant estrogen metabolism that precipitates the development of disease stages in endometriosis and/or PTB. The Braundmeier-Fleming lab has developed a systems biology model that investigates the interactions between the immune system, microbial dynamics (in the GI and reproductive system) and estrogen metabolism, in women, as a potential diagnostic tool for endometriosis and PTB. This dissertation, therefore, examined how inflammation triggered by female reproductive disorders (endometriosis or PTB) alter the systemic and localization immune responses, the microbial communities in the urogenital (UG), peritoneal and GI mucosal epithelium, as well as levels of excreted conjugated estrogen. The first specific hypothesis is that inflammation associated with endometriosis alters microbial dynamics and functions that are distinct from those of non-diseased patients. Preliminary data indicated that reproductive tract microbial communities from patients with endometriosis are unique when compared to non-disease patients. Therefore, the central aims of this study are to identify the immune and microbial profiles of patients diagnosed with endometriosis and determine if an alteration of these profiles impact estrogen signaling, thus driving disease pathogenesis. Additionally, I hypothesized that surgery or hormonal therapy will temporarily restore the microbiome and estrogen levels of patients with endometriosis. Differences in systemic (blood) regulatory T cell (Treg) and T-helper 17 (Th17) cell populations (tolerant and inflammatory, respectively) were measured by flow cytometry, and the immune mediators was measured by serum cytokine levels via 10-plex-ELISA kits. Immunohistochemistry was used to identify resident Th17/Treg immune cell distribution within the endometrium and ectopic endometriotic lesions, and RORγt+/FOXP3+ transcripts within these same tissues were analyzed by real-time-qPCR. We implemented high-throughput non genomic sequencing targeting bacterial-V4 16S rRNA and robust bioinformatics analyses to characterize microbial composition/diversity within the GI (fecal swab), vaginal (vaginal swab), and UG (urine) cavities. Alterations in estrogen metabolism, parent estrogens and metabolites, in urine were analyzed via LC-MS/MS. Patients with endometriosis exhibit 1) systemic and localized inflammation within ectopic and endometrial tissues, 2) altered GI/UG microbial dynamics, 3) aberrant levels of endogenous estrogen and estrogen metabolites, 4) dampened inflammation (caused by disease) due to hormonal therapy, 5) altered bacteria populations in the gut and vaginal canal of patients with endometriosis due to hormonal therapy treatment, and 6) increased post-surgical variability in microbial community dynamics. The second specific aims examined the hypothesis that induction of endometriosis in baboons (P. Anubis) results in chronic systemic and tissue specific inflammation through regulation of Th17 and Treg populations. Further, the induction of endometriosis altered GI/UG/peritoneal cavity microbial communities that are distinct from non-diseased animals. Utilizing a non-human primate animal model of induced endometriosis allowed us to characterize factors involved at the early onset of endometriosis and throughout the disease progression. We collected samples from 8 baboons at pre-inoculation (no evidence of disease) and at 3, 6, 9, and 15 months post-induction of the disease. We found that the induction of endometriosis decreased peripheral Tregs cells while Th17 cells increased at all post-induction collections with reduced ratio of total Tregs to Th17 cells indicating systemic inflammation. Microbial community diversities as well as abundances at each sample site (GI, UG [vagina, urine] tracts and peritoneal cavity) were also altered at post-induction. These results therefore suggest that induction of endometriosis in non-human primates caused an inflammatory shift. Disease induction also resulted in altered vaginal, urinary and fecal microbial profiles, which may drive inflammation through the production of inflammatory mediators. The last specific aims studied the hypothesis that patients who deliver preterm have a systemic and placental inflammatory phenotype and abnormal estrogen levels during pregnancy that are distinct from those of patients with term delivery. Biological samples were collected at 8-12 weeks, 20-24 weeks, 32-36 weeks, at delivery and 6 weeks postpartum. Subjects with PTB showed signs of systemic inflammation with an elevation in Th17:Treg ratio, greater Th17 and lower levels of natural Tregs during the 2nd trimester, and lower inducible Tregs during the 3rd trimester and at delivery. Placental tissues from subjects with PTB also had an inflammatory immune phenotype (higher Th17) within the decidua basalis and maternal-fetal interface. Immunological shifts from tolerant to inflammatory were observed in both patient groups, but these shifts occurred early in gestation for subjects with PTB and at a later gestational age for subjects delivering at term. Levels of conjugated parent estrogens and estrogen metabolites were reduced in subjects with PTB, indicative of an abnormal production of estrogen. These analyses gave us a better understanding of the inflammatory cascade with estrogen metabolism associated with pregnancy, and how these effects are correlated with premature labor. The data from this study suggest that the levels of endogenous estrogen and estrogen metabolites of estrogen metabolism were abnormal in PTB and endometriosis disease models of inflammation compared to their respective controls. In the human and non-human primate model of endometriosis studies, we observed that both patients and baboons with endometriosis had systemic and resident inflammatory phenotypes and an alteration in mucosal microbial community dynamics compared to their respective controls. All together, our long-term goal is to identify factors from the microbiome and/or the immune system that would allow us to have early non-invasive diagnostics for endometriosis or to predict which mothers are most at risk to encounter PTB. Furthermore, it would allow us to determine whether the mucosal microbiome may be a good indicator of immune stress, and if alternative therapies can alter microbial community dynamics—thereby eliminating immune stress associated with female reproductive diseases. These findings may have a substantial impact on the obstetrical care and management of patients with endometriosis and women at risk for PTB, as well as provide evidence to support the development of novel therapeutics to treat these diseases.

The gut microbiome and its metabolic processes are dynamic systems. Surprisingly, our understanding of gut microbiome dynamics is limited. Here we report a metaproteomic workflow that involves protein stable isotope probing (protein-SIP) and identification/quantification of partially labeled peptides. We also developed a package, which we call MetaProfiler, that corrects for false identifications and performs phylogenetic and time series analysis for the study of microbiome dynamics. From the stool sample of five mice that were fed with 15-N hydrolysate from Ralstonia eutropha, we identified 15,297 non-redundant unlabeled peptides of which 10,839 of their heavy counterparts were quantified. These peptides revealed incorporation profiles over time that were different between and within taxa, as well as between and within clusters of orthologous groups (COGs). Our study helps unravel the complex dynamics of protein synthesis and bacterial dynamics in the mouse gut microbiome.

Purpose of Review: Recent technological developments have vastly improved our ability to study the host microbiome and its role in many disease states. Numerous other reviews have contributed to our understanding of single viruses and gut microbiota or immunological outcomes. Here, we report, in aggregate, the newest data on genital microbiota interactions with the three most common viral STIs. Recent Findings: Four themes emerge: (1) the repeatability of specific community state types corresponding with infection risk, (2) a role for the microbiota as both therapeutic target and major player in treatment efficacy, (3) a need for models in which to study the mechanisms at play in microbiota/virus interactions, and (4) the impact of microbiota populating external genitalia on viral transmission. Summary: The studies reviewed herein suggest a convoluted interplay between host microbiota and viral STIs. More mechanistic studies are needed in order to leverage these interactions to improve prevention and treatment strategies.

Synthetic biology, a field that sits between Biology and Engineering disciplines, has come into its own in the last decade. The decreasing cost of DNA synthesis has lead to the creation of larger and more complex synthetic gene networks, engineered with functional goals rather than simple demonstration. While many methods have been developed to reduce the time required to produce complex networks, none focus upon the considerable tuning needed to turn structurally correct networks into functional gene networks. To this end, we created a Plug-and-Play synthetic gene network assembly that emphasizes character-driven iteration for producing functional synthetic gene networks. This platform enables post-construction modification and easy tuning of networks through its ability to swap individual parts. To demonstrate this system, we constructed a functional bistable genetic toggle and transformed it into two functionally distinct synthetic networks. Once these networks have been created and tuned at the bench, they next must be delivered to bacteria in their target environment. While this is easy for industrial applications, delivering synthetic networks as medical therapeutics has a host of problems, such as competing microbes, the host immune system, and harsh microenvironments. Therefore, we employed bacteriophage technologies to deliver functional synthetic gene networks to specific bacterial strains in various microenvironments. We first sought to deliver functional genetic networks to bacteria present in the gut microbiome. This allows for functionalization of these bacteria to eventually sense disease states and secrete therapeutics. As a proof of concept a simple circuit was created using the Plug-and-Play platform and tested before being moved into the replicative form plasmid of the M13 bacteriophage. Bacteriophage particles carrying this network were used to infect gut bacteria of mice. Infection and functionality of the synthetic network was monitored from screening fecal samples. Next, we employed phagemid technologies to deliver high copy plasmids expressing antibacterial networks to target bacteria. This allows for sustained expression of antibacterial genes that cause non-lytic bacterial death without reliance upon traditional small molecule antibiotics. Phagemid particles carrying our antibacterial networks were then tested against wild type and antibiotic-resistant bacteria in an in vitro and in vivo environment.

The composition of the gut microbiome can be affected by environmental factors, such as diet. The Western dietary pattern is associated with microbiome dysbiosis and adverse health outcomes, including obesity and metabolic disorders. The objective of this study was to examine the effect of gut microbiota from lean or obese human donors on metabolism and weight gain in recipient mice fed one of three basal diets: 1) the standard AIN93G diet, which promotes rodent health; 2) the total Western diet (TWD), which mimics the American dietary pattern and promotes inflammation-associated colorectal carcinogenesis; and 3) a 45% high fat diet-induced obesity (DIO) diet, which promotes excessive weight gain and symptoms of metabolic syndrome. We hypothesized that fecal microbiome transfer (FMT)from obese human donors would lead to an obese phenotype with symptoms of metabolic syndrome in recipient mice, and that consumption of TWD or DIO diets would further exacerbate the metabolic syndrome phenotype. The experiment design consisted of two main factors: body type of the human donor (obese or lean) and experimental diet (AIN, DIO or TWD), which was fed to mice for 22 weeks. Prior to FMT, the resident gut microbiome in mice was depleted using an established broad spectrum antibiotic/antifugal oral dosing regimen. Interestingly, human donor body type did not significantly affect final body weight or body composition in recipient

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Drought and salt stress are the main global factors that reduce the average yield of most major crops. In order to meet global demands, we will need to double food production by 2050 (Tilman, Balzer, Hill, & Befort, 2011). Plant growth-promoting bacteria (PGPB) are a group of bacteria that alleviate the harmful effects of abiotic stresses such as salt, heat and drought stress on plants and decrease the global dependence on hazardous agricultural chemicals. We identified that beneficial microbes isolated from desert plants (indigfera argentea) from Jizan region, in 2012 enhance the tolerance of a variety of crop plants to drought and salt stresses under laboratory conditions and in field trials. We analyzed the interaction of these bacteria with the plants by genetic, biochemical and imaging techniques. The goal of this dissertation is to ultimately improve our understanding of the mechanisms of drought and salt stress tolerance conferred by beneficial microbes that can be used as a sustainable solution for plants and crops in degrading lands (deserts) and land affected by abiotic stresses. Outlines how each of chapter of this dissertation will contribute to the discovery of novel drought and salt stress tolerance strategies using a desert-specific bacterial endophyte.

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No description available.