Candida albicans is an opportunistic pathogen that is present in the normal flora in a majority of individuals. One key factor in C. albicans virulence is the ability to change its morphology from yeast to an elongated or hyphal form. The regulation of this morphogenesis relies in part upon quorum sensing (QS) molecules. C. albicans often exists as part of a mixed culture alongside other microbes and is influenced by their presence as well as the presence of QS molecules that they produce. In this study, a library of diploid homozygous transcriptional regulator knockout (TRKO) mutants were screened to identify strains capable of forming hyphae in the presence of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. We identified three strains that showed increased hyphae development compared to wild type C. albicans. The strains identified had deletions of the transcriptional regulating genes Orf19.3928, Orf19.2842 (GZF3), and Orf19.3865 (RFX1). These strains were tested for alterations of filamentation in liquid media, and biofilm formation. All three strains showed increased rates of biofilm formation compared to the wild type. Orf19.3928 showed altered response to farnesol, a marked in biofilm formation and no inhibition of filamentation when farnesol was present in liquid media. The GZF3 deletion strain showed enhanced filamentation with all three bacterial species while the RFX1 deletion strain showed increased filamentation only with E. coli and S. aureus. In spent media, GZF3 showed slight increases in filamentation in E. coli and S. aureus while RFX1 had moderate increases in filamentation in E. coli and S. aureus and slight increases with P. aeruginosa.

The invasive pathways, InlA- and InB-mediated uptake and M cell transcytosis, that Listeria monocytogenes uses to invade the intestine have mainly been studied using infection models that do not truly replicate what occurs during a natural infection. Recently, our lab has developed an oral infection model that is more physiolocally relevant to what occurs during food borne listeriosis. We have sought to evaluate the relative roles of the previously defined invasive pathways, in our oral model of infection. We have done this by utilizing an InlAmCG Lm strain that is able to bind murine E-cadherin, knockout Lm strains, ΔinlA Lm, and ΔinlAΔinlB Lm. We also took advantage of a knockout mice strain CD137-/-that has M cells that are deficient in M cell transcytosis. We were able to show that these invasive pathways are relevant in our oral infection model, that M cell transcytosis is a compensatory pathway for InlA-mediated uptake, and that there might be another mechanism that L. monocytogenes uses to invade the intestines. To confirm this, it is necessary though that the M cell transcytosis deficiency be confirmed in the CD137-/- mice.

Planktothricoides raciborskii and Roseofilum reptotaenium are physiologically similar, yet ecologically distinct organisms found in a hot spring outflow and coral black band disease (BBD), respectively. The aim of this study was to elucidate the relationship between R. reptotaenium and sulfide in BBD, to compare microcystin (MC) production in response to environmental factors, and to determine chemotactic responses to MC and sulfide by the two organisms. Results showed that the pathogenicity of R. reptotaenium in BBD is dependent on sulfate-reducing bacteria as secondary pathogens. Roseofilum reptotaenium produced significantly more MC than P. raciborskii, as measured using ELISA. Roseofilum reptotaenium was negatively chemotactic to sulfide, determined using horizontal and vertical gradients in agar, while P. raciborskii was not affected. Neither cyanobacterium was chemotactic to MC in the agar assays. The ecophysiology of P. raciborskii and R. reptotaenium in relation to MC production and response to sulfide reflected their pathogenic versus non-pathogenic status.

Quorum sensing molecules have become a recent focus of study to learn if and how they can be used, both on their own and in conjecture with current antimicrobial methods, as a means of bacterial control. One such quorum sensing molecule is the sesquiterpene alcohol, Farnesol, which is synthesized and released by the fungus, Candida albicans. In most in-vivo cases, our laboratory has shown that Alcaligenes faecalis overtakes C. albicans, preventing its growth. However, as a way to counteract this inhibitory effect, Farnesol may be one way that Candida has found to fight back. In this study, we focused on the inhibitory properties of Farnesol for growth and motility of A. faecalis, as well as, the molecule’s ability to prevent Alcaligenes from creating biofilms and/or degrading them once they have already been established. Our experiments show evidence that Farnesol is able to inhibit both the growth and motility of A. faecalis, and determination of the specific concentrations of Farnesol needed to see the largest effects on A. faecalis biofilms. Our hope is that in future studies, we will be able to add varying concentrations of the Farnesol to known and widely used antibiotics in order to increase the effectiveness of antibiotics against bacterial strains, both in the Alcaligenes genus and in other genus, that have previously been considered “antibiotic resistant”.

The gut microbiota and its metabolites have vast impacts on the human digestive system, immune system, and health outcomes. Short chain volatile fatty acids (SCVFAs) present in feces can be representative of the interactions of the microbiota present in the gut. Low microbiota diversity in the human gut is highly associated with obesity and adverse health outcomes. Furthermore, the maternal microbiome has a direct impact on neonatal microbiota through various pathways such as environment, skin flora, breast milk composition, and vaginal secretions. This study is aimed to further understand the associations between various factors (maternal adiposity, gestational time, length of life, delivery mode, and race/ethnicity ) and neonatal microbiome and its metabolites, SCFA. Data (pre-pregnancy BMI, gestational time, length of life at time of sample collection, delivery mode, race/ethnicity, SCVFA profiles, fecal fermentation profiles, and 16s rRNA sequences, n=75) was obtained from 75 mother-infant dyads. Qiagen CLC Genomics Workbench was used to process 16s RNA data, generate quantitative and qualitative measures of alpha and beta diversity, and generate an analysis of the composition of microbiomes for differential abundances. Multiple metrics were analyzed for alpha and beta diversity and no significant differences were found for acetic acid (A), propionic acid (P), butyric acid (B), or APB combined. Shannon diversity index, a measure of Alpha diversity, showed no significant difference between groups in each subset. BMI differences were significant for no c-section vs. c-section and Black vs. White race/ethnicity. There were no significant differences found in PERMANOVA, a measure of beta diversity, or found in differential abundances among the groups.

Despite recent advances in antiretroviral therapy, nearly 37 million people continue to live with human immunodeficiency virus (HIV). Basic and applied research on the assembly of HIV could be enhanced by using a genetically tractable organism, such as yeast, rather than mammalian cells. While previous studies showed that expression of the HIV Gag polyprotein in Saccharomyces cerevisiae spheroplasts resulted in the production of virus-like particles (VLPs), many questions regarding the utility of yeast in HIV assembly remain uninvestigated. Here, we report use of S. cerevisiae for both the production of VLPs with selectively packaged RNA and to evaluate the human Y-box-binding protein 1 (YB-1) in selective RNA packaging into VLPs. Our data reveal: (1) When co-expressed alongside HIV-1 Gag, an RNA mammalian expression cassette is selectively encapsidated and released in VLPs produced from spheroplasts; (2) Inclusion of the 5’UTR-5’Gag RNA upstream of the mammalian expression cassette greatly increased the selectivity to which non-viral RNA was packaged into VLPs; and (3) heterologous expression of the human YB-1 protein in S. cerevisiae did not facilitate the selective packaging of viral RNA into VLPs, likely due to inability to bind upstream elements in the HIV-1 viral RNA. Overall, this research provides a key first step in the use of yeast for the production of viral vectors used in gene therapy, and lays a foundation for further experiments investigating the role of YB-1 and other host proteins in selective RNA packaging.

For several decades amphibian populations have been declining. Historically, the bacterium A. hydrophila (Ah) was hypothesized to be the causal factor in amphibian disease and population declines. However, with the discovery of a chytrid fungus, Batrachochytrium dendrobatidis (Bd) in 1998, which was identified on the skin of amphibians during documented mortality events, Ah research became of minor interest as focus shifted to Bd. Recent studies into the immunocompromising abilities of Bd, however, have opened new questions about its relationship with Ah and their combined effects on a host. In this study, I explore the relationship between infection with these two pathogens, Bd and Ah, in two amphibian species from distinct regions of the United States. I developed a novel qPCR assay to measure the microbial load of Ah on the skin of two anuran species, Lithobates yavapaiensis (N=232) and Pseudacris ornata (N=169), which have confirmed Bd infections. I use a logistic regression model to identify whether significant relationships exist between these two pathogens, disease, and death. I find that even amongst the most severely infected frogs, Ah is not detectable on the skin and only appears post-mortem. I therefore conclude that Ah is an opportunistic bacterial pathogen, scavenging on anurans only after mortality events. This research is the first known study to quantitatively assess Ah in amphibians in conjunction with Bd. While there is no causal relationship between these pathogens, future work will examine potential Ah infections in other organs to more fully understand the relationship between Bd and Ah.

We have previously demonstrated that long-term dietary Se deficiency in old Terc-/- mice with humanized telomeres induces type-2 diabetes and exacerbates age-dependent increases in the abundance of A. muciniphila and Lachnospiraceae, which are related to obesity and metabolic syndromes. The objectives of this dissertation are: 1) to determine the minimum intake of Se required for type 2 diabetes prevention in middle-aged mice; 2) to evaluate the efficacy of A. muciniphila and R. torques (a Lachnospiraceae family member) to intervene dietary Se deficiency-induced type 2 diabetes and the underlying mechanisms; 3) to assess sex differences in the responses to dietary Se deficiency and oral gavage of such bacteria. Our results demonstrated that mice fed diets containing ≤0.10 mg Se/kg developed glucose intolerance and insulin resistance at middle-aged stage. To address objectives 2 and 3, we showed that dietary Se deficiency exacerbated type-2 diabetes-like phenotypes in males but the extent was less in females aged 7 and 13 months. Oral gavage of A. muciniphila into either antibiotics-treated or conventional mice ameliorated these phenotypes and elevated beneficial bacteria (Lactobacillus, F. prausnitzii, and Roseburia spp./E. rectale) abundance, but reduced E. coli abundance. Dietary Se deficiency decreased intestinal barrier functions and induced intestinal inflammation. In conventional mice, A. muciniphila oral gavage reversed such intestinal defects but did not affect the expression of selenoproteins. By contrast, oral gavage of R. torques did not restore dietary Se deficiency-induced type 2 diabetes-like phenotypes in female mature mice and showed opposite impacts on the change of the 4 specific genera in comparison with A. muciniphila oral gavage. Taken together, our findings demonstrate that suboptimal body Se status induces type 2 diabetes and reshapes gut microbiota in an age- and sex-dependent manner. Such metabolic defects in conventional Se-deficient mice can be alleviated by A. muciniphila but not R. torques supplement, which may counteract common intestinal defects in metabolic syndrome. In conclusion, optimal Se at nutritional level of intake is necessary to prevent type 2 diabetes. A. muciniphila is a promising supplement for alleviation of type 2 diabetes and possibly other metabolic diseases in relation to intestinal inflammation and glucose dysregulation.

In this study, Naegleria fowleri, a pathogenic amoeba and the causative agent of Primary Amebic Meningoencephalitis (PAM), was utilized to determine the presence or absence of classically conserved Golgi molecules featured in the expression of a Golgi apparatus. Previous studies concluded no Golgi expression via light microscopy and transmission electron microscopy, but a recent report on Naegleria gruberi indicated the presence of dispersed Golgi tubules. Non-pathogenic species of the Naegleria genus such as Naegleria gruberi 30540 and Naegleria lovaniensis 30569 were utilized in Western immunoblot analysis compared to reduced whole-cell lysate proteins of two strains of N. fowleri and Vero CCL-81, Chlorocebus sp. kidney epithelial cells, which were utilized as a positive control for Golgi expression. N. fowleri and N. lovaniensis whole-cell lysates had indications of a 110 kDa reduced protein, associated with the predicted molecular weights of the beta-COPI subunit of the COPI cis-Golgi vesicular transport complex with further Western immunoblot indication of a weak band around 25 kDa corresponding to rabbit polyclonal antibodies specific for ARF1. Serial Dilutions of Wheat Germ Agglutinin Alexa Fluor 488TM were performed on Vero cells, Naegleria fowleri 30894, and N. gruberi 30540 with 1:100 dilution of recommended stock dilution of WGA 488 determined for utilization in sequential immunofluorescence. Sequential immunofluorescence with Wheat Germ Agglutinin Alexa Fluor 488TM and then blocked with 3% BSA:PBS [wt/vol] dilution with subsequent incubation in rabbit anti-beta-COPI primary 1:250, and 1:1000 of Alexa Fluor 594 goat anti-rabbit secondary antibody exposure showed strong indications of organized cis- and trans-punctate Golgi body markers in close association in individual and dividing cells of Naegleria fowleri and conserved Golgi expression in the positive control Vero cells, but further experiments are necessary to verify this finding with N. fowleri.

Breast cancer mortality is mainly due to distant recurrence of the disease arising from dormant tumor cells established by cancer therapies. Patients who initially respond to cancer therapies often succumb to distant recurrence of the disease. It is not clear why people with the same type of breast cancer respond to treatments differently; some escape from dormancy and relapse earlier than others. In addition, some tumor clones respond to immunotherapy while others do not. We investigated how autophagy plays a role in accelerating or delaying recurrence of neu overexpressing mouse mammary carcinoma (MMC) following adriamycin (ADR) treatment, and in affecting response to immunotherapy. We explored two strategies: 1) transient blockade of autophagy with chloroquine (CQ), which blocks fusion of autophagosomes and lysosomes during ADR treatment, and 2) permanent inhibition of autophagy by a stable knockdown of ATG5 (ATG5KD), which inhibits the formation of autophagosomes in MMC during and after ADR treatment. We found that while CQ prolonged tumor dormancy, but that stable knockdown of autophagy resulted in early escape from dormancy and recurrence. Interestingly, ATG5KD MMC contained an increased frequency of ADR-induced polyploid-like cells and rendered MMC resistant to immunotherapy. On the other hand, a transient blockade of autophagy did not affect the sensitivity of MMC to immunotherapy. Our observations suggest that while chemotherapy-induced autophagy may facilitate tumor relapse, cell-intrinsic autophagy delays tumor relapse, in part, by inhibiting the formation of polyploid-like tumor dormancy. Although immunotherapy of breast cancer by means of anti-HER2 antibodies prolongs survival of breast cancer patients, disease recurrence remains a major challenge. On the other hand administration of human vaccines against infectious disease in a preventive setting or during latency/dormancy has been successful in offering a cure. Here, we sought to use adoptive immunotherapy (AIT) at the time of tumor dormancy in order to prevent progression of breast cancer. We used a low dose immunogenic chemotherapy by means of 5-FU, Adriamycin, and Cyclophosphamide (FAC) in order to stabilize tumor progression prior to AIT using autologous tumor-reactive lymphocytes. Low dose FAC established local tumor dormancy, inhibited distant tumor dormancy occurring long before distant metastasis, and induced predominate a Ki67- quiescent type of tumor dormancy, which is less susceptible to tumor immunoediting. Dormant tumor cells expressed the cell survival pathways, including the endothelin receptor/ligand (ETRA, ETRB and ET-1) and PD-L1, thereby protecting them from elimination by AIT. In addition, tumor-reactive CD8+ T cells also produced ET-1 as a survival ligand for ETRA positive tumor cells. A combination of AIT with the blockade of tumor cell survival pathways resulted in a significant improvement of AIT against tumor dormancy. We also showed that the inhibition Bcl-xL downstream of the tumor cell survival pathways is specifically effective against dormant tumor cells, suggesting a combination of AIT with small molecules inhibitors of Bcl-xL. Altogether, we showed that distant tumor dormancy is established long before distant recurrence of breast cancer, and that the expression of several tumor cell survival pathways in dormant cells protects them from immunotherapy. Our results suggest that immunotherapeutic targeting of tumor dormancy combined with the blockade of a common downstream cell survival pathway could prevent tumor progression and recurrence of the disease.