Intestinal epithelial cells function, in part, to detect infection with pathogenic organisms and are key regulators of intestinal immune homeostasis. However, it is not fully understood how intestinal epithelial cells sense pathogen infection and coordinate the induction of protective immune defenses. Here, we define two new mechanisms of innate immune regulation in a metazoan host. First, we characterize the first bacterial pattern recognition receptor and its natural ligand in Caenorhabditis elegans. We show that the C. elegans nuclear hormone receptor NHR-86/HNF4 directly senses phenazine-1-carboxamide (PCN), a metabolite produced by pathogenic strains of Pseudomonas aeruginosa. PCN binds to the ligand-binding domain of NHR-86/HNF4, a ligand-gated transcription factor, and activates innate immunity in intestinal epithelial cells. In addition, we show that C. elegans NHR-86 senses PCN, and not other phenazine metabolites, as a marker of pathogen virulence to engage protective anti-pathogen defenses. Second, we show that a phase transition of the C. elegans Toll/interleukin-1 receptor domain protein (TIR-1) controls signaling by the C. elegans p38 PMK-1 MAPK pathway. Physiologic stress, both P. aeruginosa infection and sterol scarcity, induce multimerization of TIR-1 within intestinal epithelial cells. Like the mammalian homolog of TIR-1, SARM1, oligomerization and phase transition of C. elegans TIR-1 dramatically potentiate its NAD+ glycohydrolase activity. TIR-1/SARM1 multimerization and NAD+ glycohydrolase activity are required for activation of C. elegans p38 PMK-1 pathway signaling and pathogen resistance. These data uncover a mechanism by which nematodes interpret environmental conditions to prime innate immune defenses and promote survival in microbe rich environments. C. elegans animals augment these immune defenses by surveying for ligands specifically associated with toxigenic pathogens that are poised to cause disease. These findings define a new paradigm of intestinal immune control that informs the evolution of innate immunity in all metazoans.

Avian pathogenic Escherichia coli (APEC) is a bacterium that is responsible for colibacillosis in birds. However, information about broiler breeder APEC isolates is limited, but the data is critical due to the transfer of this bacteria down the production pyramid to progenies resulting in high mortality. Therefore, we evaluated the phenotypic virulence characteristics of 28 isolates using embryo lethality and day-old chick challenge assays. Also, the in vitro adhesion and invasion potential of selected nine isolates were identified. Results showed more than 1/3rd of the isolates were highly virulent and the virulence increased as the number of virulence-associated genes increased. High adhesion and invasion rates were observed among the isolates. Overall, the study helped us to evaluate the virulence characteristics of APEC from broiler breeders. However, future studies based on whole genome approach would help to identify the specific targets which can be used to develop effective interventions.

Myeloid-derived suppressor cells (MDSCs) numbers increase significantly in sepsis and are associated with high mortality rates. These myeloid cell precursors promote immunosuppression, especially in the late (post sepsis) stage. However, the mechanisms that underlie MDSC expansion and programming are not completely understood. To investigate these mechanisms, we used a cecal-ligation and puncture (CLP) mouse model of polymicrobial sepsis that progresses from an early/acute proinflammatory phase to a late/chronic immunosuppressive phase. Previous studies in our laboratory showed that microRNA (miR)-21 and miR-181b elevate levels of the transcription factor nuclear factor 1 (NFI-A) that promotes MDSC expansion. We report here that miR-21 and miR-181b regulate NFI-A expression via a post-transcriptional regulatory mechanism by recruiting RNA-binding proteins HuR and Ago1 to stabilize NFI-A mRNA, thus increasing its protein levels. Studies in our laboratory also showed that inflammatory mediator S100A9 accumulates in the nucleus in Gr1+CD11b+ myeloid precursors in the later phases of sepsis and is necessary for their expansion and programming into immunosuppressive MDSCs. We demonstrate here that nuclear S100A9 associates with specific transcription factors that activate miR-21 and miR-181b expressions. In our final manuscript, we uncover another layer of the mechanisms of MDSC expansion and programming. We found that long non-coding RNA (lncRNA) Hotairm1 binds to and recruits S100A9 to the nucleus to program Gr1+CD11b+ myeloid precursors into MDSCs in the later phases of sepsis. Together, our results reveal three regulatory layers involving NFI-A, S100A9 and Hotairm1 in the pathway leading to MDSCs development in sepsis and suggest that therapeutically targeting these molecular switches might improve sepsis survival.

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAO∆ampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.

Neisseria gonorrhoeae (Ng) which causes gonorrhea has become multidrug-resistant, necessitating the development of novel therapeutics and vaccines. mAb 2C7 which targets an epitope within an important virulence factor, the lipooligosaccharide (LOS), is a candidate therapeutic mAb. Ninety-four percent of clinical isolates express the 2C7-epitope which is also a vaccine target. Ng expresses multiple LOS(s) due to phase-variation (pv) of LOS glycosyltransferase (lgt) genes. mAb 2C7 reactivity requires a lactose extension from the LOS core Heptose (Hep) II (i.e. lgtG ‘ON’ [G+]). Pv results in HepI with: two (2-), three (3-), four (4-), or five (5-) hexoses (Hex). How HepI glycans impact Ng infectivity and mAb 2C7 function are unknown and form the bases of this dissertation. Using isogenic mutants, I demonstrate that HepI LOS glycans modulate mAb 2C7 binding. mAb 2C7 causes complement (C’)-dependent bacteriolysis of three (2-Hex/G+, 4-Hex/G+, and 5-Hex/G+) of the HepI mutants in vitro. The 3-Hex/G+ mutant (resistant to C’-dependent bacteriolysis) is killed by neutrophils in the presence of mAb and C’. In mice, 2- and 3-Hex/G+ infections are significantly shorter than 4- and 5-Hex/G+ infections. A chimeric mAb 2C7 that hyperactivates C’, attenuates only 4- and 5-Hex/G+ infections. This study enhances understanding of the role of HepI LOS pv in gonococcal infections and shows that longer HepI glycans are necessary for prolonged infections in vivo. This is the first study that predicts in vitro efficacy of mAb 2C7 against all four targetable HepI glycans thereby strengthening the rationale for development of 2C7-epitope based vaccines and therapeutics.

Lilium grayi (Gray’s Lily), a southern Appalachian endemic species, is threatened by a Lilium-specific fungal pathogen, Pseudocercosporella inconspicua. The disease is characterized by tan lesions that can cause early senescence, while also lowering seed production and viability. This project tested for P. inconspicua conidia and accessed health at nine locations. The disease was present and ubiquitous across the range of L. grayi. Through identification of P. inconspicua conidia in the field, L. superbum (Turk’s Cap Lily) was identified as an additional host, while L. michauxii (Michaux’s Lily) was disease-free. However, infection was inducible in both species. With the disease widespread in L. superbum and this species represented by many large populations, L. superbum may act as disease reservoir, further complicating the outlook for L. grayi. The disease should be considered an epidemic because of its impact on individual plants, its commonness within populations, and its ubiquity across the geographical range.

Food borne illnesses continues to be a public health challenge in the United States (U.S.); an estimated 9.4 million incident cases occurred in 2011. In view of this challenge we conducted two food safety studies; 1) related to product formulation (hummus spoilage challenge study) and 2) evaluating the microbial safety of domestic kitchen surfaces in Residential Child Care Institutions (RCCI pilot study). Hummus is of Mediterranean origin but is currently eaten globally. This challenge study evaluates a variety of industrial hummus formulations (four in total, differing in pH and/or addition of a preservative (natamycin). Two batches were setup: batch 1; aseptically inoculated hummus with 100 CFU/g fungal isolates and batch 2; uninoculated hummus. Samples of both hummus batches were stored at both 20oC (10 days accelerated testing) and 4oC (84 days recommended temperature testing). Inoculated samples were analyzed for fungus, whiles both fungi and bacteria (standard plate count (SPC) and Lactococci) counts were done for uninoculated samples. Results indicate that accelerated testing inaccurately predicts fungal growth at 4oC in hummus, also fungal growth inhibition requires a pH ≤ 4.0 ± 0.2 and refrigeration. Limited studies have specifically evaluated the prevalence of pathogenic bacteria in domestic kitchens in the U.S, for this reason we assessed the microbial safety of 6 RCCI locations in MA. Fifteen key food contact surfaces and dish washing sponges, if available at each RCCI facility were assessed for SPC, yeast and molds, total coliform and E. coli, Listeria sp and Salmonella sp. Microbiological assessments were conducted preceding and after a hazard analysis and critical control point (HACCP) food safety training and implementation at each location. Microbial growth varied by surface for each type of microorganism, wet surfaces had higher most probable number (MPN) counts. Compared to dry surfaces, wet surfaces had significantly higher mean total coliform counts. For both E. coli and total coliform, microbial load differed significantly by surfaces sampled (P = 0.0323 and 0.014) respectively. The surface and training interaction effect was highly significant for only E. coli (P = 0.0089). Training overall had no significant effect on reducing the microbial load on kitchen surfaces.

Packed-bed digesters are an alternative to covered lagoon digesters for methane production and anaerobic treatment of dilute wastewaters such as dairy barn flush water. The physical media of packed-beds retain biofilms, often allowing increased treatment rates. Previous studies have evaluated several types of media for digestion of dilute wastewaters, but cost and media fouling have setback commercial development. A major operational cost has been effluent recirculation pumping. In the present effort, a novel approach to anaerobic digestion of flush dairy water was developed at pilot-scale: broken walnut shells were used as a low-cost packed-bed medium and effluent recirculation was replaced by reciprocation mixing to decrease pumping costs and the risk of media clogging. Three packed-bed digesters containing walnut shells as media were constructed at the on-campus dairy and studied for about six months. Over that time, several organic loading rates (OLRs), measured as both chemical oxygen demand (COD) and volatile solids (VS) were applied to the new packed-bed digesters to allow modeling of methane production. The influence of temperature on methane production was also investigated. Additionally, the study measured solids accumulation in the walnut shell packed-bed as well as the effectiveness and durability of walnut shells as packing media. Finally, a simple economic analysis was developed from the methane model to predict the financial feasibility of packed-bed digesters at flush water dairies under similar OLR conditions. Three methane production models were developed from organic loading: saturation-type (following the form of the Monod equation), power and linear. The models were evaluated in terms of regression analysis and the linearity of experimental to predicted methane production. The best model was then chosen to develop the economic predictions. Economic predictions for packed-bed digesters were calculated as internal rate of return (IRR) using the methane models along with additional input variables. Comparisons of IRRs were made using electric retail rates of $0.10 to $0.20 per kilowatt-hour and capital cost subsidies from zero to 50%. Sludge accumulation in the packed-bed was measured via change in porosity, and walnut shell durability was measured as the change in mass of representative walnut shells over the course of the study. The linear-type model of methane production from volatile solids OLR best represented this data set. Digester temperature was not found to influence methane production in this study, likely due to the small daily average ambient temperature range experienced (14°C to 24°C) and the greater influence of organic loading. Porosity of the walnut shell packed-bed decreased from 0.70 at startup to 0.34±0.06 at the end of the six-month study, indicating considerable media fouling. Sludge accumulated in each digester from zero at startup to 281±46 liters at termination. Walnut shells in the packed-bed lost on average 31.4±6.3% mass during the study period which may be attributed to degradation of more readily bio-degradable cellulose and hemi-cellulose within the walnut shells. Given the predicted methane production and media life, at present, the economic outlook for packed-bed digesters at commercial dairies is quite dependent on utility electrical rates, available subsidies and future improvements to packed-bed digester technology. The predicted IRRs ranged from below 0% (at 0% capital subsidy and $0.10/kWh) up to 25% (at 50% capital subsidy and $0.20/kWh) at large dairies (3000 milking cows). Increases in organic loading were not shown to necessarily increase IRR, particularly at OLRs above 10 g/Lliquid-d (as COD or VS). Ultimately, to better assess the value of packed-bed digesters for flush dairies, additional study is needed on topics such as sludge accumulation prevention, long-term walnut shell degradation, dairy barn flush water mixing, and more detailed economic analysis.

Human body lice, Pediculus humanus humanus, and head lice, Pediculus humanus capitis, have been hematophagous ectoparasites of humans for thousands of years. Despite being ecotypes, only body lice are known to transmit bacterial diseases to humans, and it appears that lower humoral and cellular immune responses allow body lice to possess a higher vector competence. We previously observed that the transcription level of the defensin 1 gene was up-regulated only in head lice following oral challenge of Bartonella quintana, a causative agent of trench fever, and also that body lice excreted more viable B. quintana in their feces. In this study, we first investigated this differential immune response by performing RNAi to knockdown defensin 1 by dsRNA injection. B. quintana was orally infected 72 h after injection and proliferation was compared at 2 hours (day 0) and day 4 post-infection. At day 0, bacterial cell numbers increased 1.5-fold in defensin 1 (Def1(-)) knocked down head lice compared with non-knocked down, pQE30-dsRNA injected, head lice control. At day 4, Def1(-) knocked down head lice had 2.55-fold more bacterial cells than control head lice and 1.65-fold greater than body lice, indicating that defensin 1 was active in reducing B. quintana cell number in non-knocked down head lice. Second, the levels of cytotoxic reactive oxygen species (ROS) generated by the epithelial cells of the alimentary tract were measured using two general indictors of ROS in both body and head lice at day 1 and day 4 following B. quintana challenge. Challenged body lice showed a 42% and 34% increase in ROS, whereas head lice showed a 70% and 22% increase at day 1 using CM-H2DCFDA and HPF as general indicators, respectively. On day 4, all challenged lice showed similar ROS levels except for body lice which maintained their ROS levels (40% increase using CM-H2DCFDA). Head lice are likely to have multiple immune and/or non-immune factors that suppress B. quintana proliferation, and the production of sustained ROS levels and/or the single knockdown of Defensin 1 is not enough to increase B. quintana proliferation in head lice to that seen in body lice.