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.

Streptococcus zooepidemicus (Sz) and Streptococcus equi (Se) share 98% DNA sequence homology, but display different pathogenic properties. Infection by one organism does not cross-protect against the other. To better understand pathogenic differences between these organisms and gain information about which proteins are expressed in horses infected experimentally with Se, intrauterine Sz or naturally with respiratory Sz we compared antibody specificities of convalescent sera using ELISA. These comparisons were based on sets of 8 and 14 immunoreactive recombinant proteins of Se strain CF32 and Sz strain NC78, respectively. Sera from donkeys that were previously naturally affected with strangles and later developed Sz pneumonia secondary to an experimental influenza challenge were also included. Serum antibody responses were quantitatively and qualitatively much greater following recovery from strangles than following respiratory Sz infection. Increased reactions to Se proteins IdeE2, Se75.3, Se46.8, Se18.9 and Se42.0 were observed for the majority of strangles sera but not for sera from respiratory Sz infection cases. Reactions of sera from Sz respiratory disease to Sz proteins varied greatly and were mostly to HylC and ScpC. Interestingly, sera of donkey recovered from Sz bronchopneumonia did not show increased antibody reaction to any of the proteins even though these donkeys had also recovered from clinical strangles 6 months previously. Only 1/5 mare with Sz placentitis presented increased serum antibody responses to MAP. In conclusion, adaptive immune responses to Se of horses with strangles are stronger and involve a greater number of proteins than adaptive immune responses to Sz infection of the lower respiratory tract. In an effort to develop an improved vaccine against Se, modified live strain of EHV-1, RacH was constructed to express three recombinant antigens of Se SeM, IdeE and Se18.9. Two groups of 10 and 2 ponies were vaccinated intramuscularly or intranasally, respectively. Another group (n=6) vaccinated with empty RacH served as controls. Sera from 2/3 ponies from each vaccination groups and 1/2 serum from IN vaccinated ponies showed increased serum neutralizing antibodies to EHV-1. ELISA detected no significant increase in antibodies to proteins. Only one IM and IN vaccinated pony showed serum bactericidal activity post vaccination.

Black band disease (BBD) of corals is a temperature dependent, highly virulent, polymicrobial disease affecting reef-building corals globally. The microbial consortium of BBD is primarily comprised of functional physiological groups that include photosynthetic cyanobacteria, sulfate reducers, sulfide oxidizers and a vast repertoire of heterotrophic bacteria. Quorum sensing (QS), the cell-density dependent communication phenomenon in bacteria, is known to induce expression of genes for a variety of virulence factors in diseases worldwide. Microbes capable of QS release signals such as acyl homoserine lactones (AHLs) and autoinducer-2 (AI-2), which coordinate microbial interaction. The focus of the present study was to investigate the presence and potential role of QS in BBD pathogenicity, utilizing culture dependent and independent methodologies. Isolates across coral health states including BBD, were screened for production of QS signals, and AHL and AI-2 production capabilities were analyzed via LC-MS/MS. The effect of temperature on AHLs was also examined. Additionally, antimicrobial production capabilities of isolates were tested. BBD metagenomes were utilized to screen for sequences related to QS, antimicrobial synthesis, and antimicrobial resistance genes. BBD isolates represented a significantly higher proportion of isolates capable of producing QS signals in comparison to healthy coral isolates. Several AHLs produced by coral derived bacterial cultures were identified, and three AHLs, specifically 3OHC4, 3OHC5 and 3OHC6, showed a significant increase in production at an elevated temperature of 30 °C, which correlates with increased BBD incidence on reefs with increasing water temperature. Most of the BBD cultured isolates were identified as vibrios. Several sequences related to QS, antimicrobial synthesis and resistance genes were detected in the BBD metagenomes. Based on the findings of this study, a model for potential microbial interactions amongst BBD heterotrophs, centered around QS, is proposed. Taken together, the findings from this study provide a clearer understanding of the potential role of QS in BBD, and serve as the basis for further studies aimed at elucidating the pathogenesis of an intricate coral disease.

Coral reefs are one of the most diverse ecosystems on the planet due partially to the habitat structure provided by corals. Corals are long lived organisms that can live for hundreds of years and as a result growth of many species is very slow. As a result of this, recovery of corals from disease outbreaks is very slow and difficult and therefore the ecosystem is deteriorating rapidly. Due to this increase in disease and its detrimental effect on coral reefs, it has become imperative to study how corals respond to disease outbreaks. The response of the coral to pathogens is believed to be controlled by the innate immune system. However, the immune pathways and components of these pathways used by cnidarians to combat pathogens are still rudimentary. This work showed that C3 and heat shock protein 70 are components of the coral immune system that positively respond to disease occurrence. As disease out breaks become more frequent, the question has arisen as to whether cnidarians have homologs to of the adaptive immune system that allow them to respond more rapidly to subsequent encounters with the same bacterium. In the cnidarian model system Exaiptasia pallida, immune priming occurs up to one month after the initial sub lethal exposure to the pathogen. This transient form of priming could be the result of host energy allocation in place of establishing long term immune priming which could be too energetically costly. Cnidarians may only activate priming during summer months, when ocean temperatures and bacterial load are high. Specificity of immune priming in E. pallida requires further investigation with more bacterial pathogens. In this dissertation, one bacterial strain shows specificity while the other does not. Furthermore, the priming response involves many pathways which include pathogen recognition, inflammation, and activation of NF-κB. The discovery of immune priming in a sea anemone shows that this phenomenon evolved earlier in the tree of life than previously thought. Additionally, identification of priming in E. pallida is suggestive of its presence in corals which would allow for potential vaccinations of vulnerable corals.

Pseudomonas aeruginosa is a Gram-negative, metabolically versatile, opportunistic pathogen that exhibits a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability is in part mediated by two-component systems (TCS) that play a crucial role in regulating virulence mechanisms, metabolism and antibiotic resistance. Our sequence analysis of the P. aeruginosa PAO1 genome revealed the presence of two open reading frames, mifS and mifR, which encodes putative TCS proteins, a histidine sensor kinase MifS and a response regulator MifR, respectively. This two-gene operon was found immediately upstream of the poxAB operon, where poxB encodes a chromosomal ß-lactamase, hinting at the role of MifSR TCS in regulating antibiotic resistance. However, loss of mifSR had no effect on the antibiotic resistance profile when compared to P. aeruginosa parent PAO1 strain. Subsequently, our phenotypic microarray data (BioLOG) and growth profile studies indicated the inability of mifSR mutants to grow in α-ketoglutarate (α-KG), a key tricarboxylic acid (TCA) cycle intermediate, as a sole carbon source. To date, very little is known about the physiology of P. aeruginosa when provided with α-KG as its sole carbon source and the role of MifS and MifR TCS in virulence. Importantly, in the recent years, α-KG has gained notoriety for its newly identified role as a signaling molecule in addition to its conventional role in metabolism. This led us to hypothesize that MifSR TCS is involved in α-KG utilization and virulence in P. aeruginosa. Using mifS, mifR and mifSR clean in-frame deletion strains, our study demonstrates that the MifSR TCS modulates the expression P. aeruginosa kgtP (PA5530) and pcaT (PA0229) genes encoding putative α-KG permeases. In addition, our study shows that the MifSR-regulation of these transporters requires functional sigma factor RpoN (σ54). Loss of mifSR in the presence of α-KG, resulted in differential regulation of P. aeruginosa key virulence determinants including biofilm formation, motility, cell cytoxicity and the production of pyocyanin and pyoverdine. Involvement of multiple regulators and transporters suggests the presence of an intricate circuitry in the transport of α-KG and its importance in P. aeruginosa survival. This is further supported by the α-KG-dependent MifSR regulation of multiple virulence mechanisms. Simultaneous regulation of multiple mechanisms involved in P. aeruginosa pathogenesis suggests a complex mechanism of MifSR action. Understanding the physiological cues and regulation would provide a better stratagem to fight often indomitable P. aeruginosa infections.

Klebsiella pneumoniae is a Gram-negative pathogen associated with numerous infections. Like all Gram-negative bacteria, K. pneumoniae naturally release outer membrane vesicles (OMVs) during all stages of cellular growth. OMVs are composed of the outer membrane components such as lipopolysaccharide (LPS) and outer membrane proteins and contain cytosolic and periplasmic proteins in the lumen. K. pneumoniae is often found to lack an O antigen. The absence of the O antigen has been reported to alter the protein content of the membrane which may further alter the immune response elicited by K. pneumoniae. Therefore the purpose of this study was to analyze the protein content and inflammatory responses of the cellular outer membrane (OM) and the secreted OMVs. A secondary goal of this study was to evaluate any changes to the membrane protein content due to O antigen loss and determine if the loss of the O antigen influences the inflammatory response. This study demonstrated that the cellular OM and OMVs have distinct protein profiles as well as inflammatory responses. OMVs were highly enriched in outer membrane proteins compared to the cellular OM and had a significantly stronger inflammatory response. The protein content of the OM and OMVs were further modified by the absence of the O antigen from the outer membrane. Although there were no significant differences seen in the wild type and O antigen deficient OM inflammatory responses, the loss of the O antigen resulted in OMVs that produced a significantly stronger IL-6 response.

Clarireedia spp. (formerly Sclerotinia homoeocarpaF.T. Bennett) is the causal agent dollar spot, the most economically important turfgrass disease impacting golf courses in North America. The most effective strategy for dollar spot control is repeated application of multiple classes of fungicides. However, reliance on chemical application has led to resistance to four classes of fungicides as well as multidrug resistance (MDR). Fungi are known to detoxify xenobiotics, like fungicides, through transcriptional regulation of three detoxification phases: modification, conjugation and secretion. Little is known, however, of the protein-protein interactions that facilitate these pathways. Following next-generation RNA sequencing of Clarireedia spp., a fungus-specific transcription factor, XDR1, was determined to play a role in constitutive and induced overexpression of phases I and III genes of xenobiotic detoxification. Further, a novel activation domain (AD) on XDR1 that does not directly bind with xenobiotics was confirmed to be highly conserved among fungal species. Therefore, we hypothesize that XDR1 must be activated by interacting with other binding partners at this AD in order to regulate downstream xenobiotic detoxification pathways. The main objective of this study is to identify additional proteins/ co-repressors that activate XDR1 in order to gain a better understanding of how transcriptional regulation of xenobiotic detoxification pathways leads to MDR. In order to test the hypothesis, fungicide sensitive strain (HRS10) and fungicide resistant strain (HRI11) were transformed and tagged with xdr1/XDR1 and the 3xFLAG tag. As a result, four fungal transformants were generated and those are HRS10-XDR1-3xFLAG, HRS10-xdr1- 3xFLAG, HRI11-XDR1-3xFLAG, and HRI11-xdr1-3xFLAG. The total protein extractions (whole cell lysates) were subjected to co-immunoprecipitation and the samples were analyzed using LC-MS/MS. According to the set of results, more than 50 proteins were detected with HRS10-XDR1-3xFLAG with and most of these binding partners having functions related to post translational modification, protein turnover, intracellular trafficking, secretion and vascular transport. Going forward, information gained from this experiment could be used to explore how XDR1 interacts with its binding partners to facilitate the transcription of drug metabolizing genes responsible for multidrug resistance. This information could also help identify additional fungicide metabolism pathways in filamentous fungi.

Variation in the sequence of T cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T cell responses during second heterologous infections contributing to pathology following DENV infection. We identified a highly conserved, novel, HLA-B57-restricted epitope on the DENV NS1 protein, NS126-34. We predicted higher frequencies of NS126-34-specific CD8+ T cells in PBMC from individuals undergoing secondary, rather than primary, DENV infection due to the expansion of memory CD8+T cells. We generated a tetramer against this epitope (B57-NS126-34TET) and used it to assess the frequencies and phenotype of antigen-specific T cells in samples from a clinical cohort of children with acute DENV infection established in Bangkok, Thailand. High tetramer-positive T cell frequencies during acute infection were seen in only 1 of 9 subjects with secondary infection. B57-NS126-34-specific, other DENV epitope-specific CD8+ T cells, as well as total CD8+ T cells, expressed an activated phenotype (CD69+ and/or CD38+) during acute infection. In contrast, expression of CD71 was largely limited to DENV-specific CD8+ T cells. In vitro stimulation of CD8+ T cell lines, generated against three different DENV epitopes, indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides with substantial upregulation of CD71 detected to peptides which also elicited strong functional responses. CD71 may therefore represent a useful marker of antigenspecific T cell activation. During the course of our analysis we found substantial binding of B57-NS126-34 TET to CD8- cells. We demonstrated that the B57-NS126-34 TET bound KIR3DL1, an inhibitory receptor on natural killer (NK) cells. NK sensitive target cells presenting the NS126-34 peptide in the context of HLA-B57 were able to dampen functional responses of only KIR3DL1+ NK cells. Analysis of the activation of an NK enriched population in our Thai cohort revealed peak activation during the critical time phase in patients with severe dengue illness, dengue hemorrhagic fever, compared to people with mild illness. Our data identified CD71 as biologically useful marker to study DENV-specific CD8+ T cell responses and highlighted the role of viral peptides in modulating NK cell activation through KIR-MHC class I interactions during DENV infection.

Salmonella enterica subtype Typhimurium (S. Typhimurium) is one of many non-typhoidal Salmonella enterica strains responsible for over one million cases of salmonellosis in the United States each year. These Salmonella strains are also a leading cause of diarrheal disease in developing countries. Nontyphoidal salmonellosis induces gastrointestinal distress that is characterized histopathologically by an influx of polymorphonuclear leukocytes (PMNs), the non-specific effects of which lead to tissue damage and contribute to diarrhea. Prior studies from our lab have demonstrated that the type III secreted bacterial effector SipA is a key regulator of PMN influx during S. Typhimurium infection and that its activity requires processing by caspase-3. Although we established caspase-3 activity is required for the activation of inflammatory pathways during S. Typhimurium infection, the mechanisms by which caspase-3 is activated remain incompletely understood. Most challenging is the fact that SipA is responsible for activating caspase-3, which begs the question of how SipA can activate an enzyme it requires for its own activity. In the present study, we describe our findings that the eukaryotic tetraspanning membrane protein PERP is required for the S. Typhimuriuminduced influx of PMNs. We further show that S. Typhimurium infection induces PERP accumulation at the apical surface of polarized colonic epithelial cells, and that this accumulation requires SipA. Strikingly, PERP accumulation occurs in the absence of caspase-3 processing of SipA, which is the first time we have shown SipA mediates a cellular event without first requiring caspase-3 processing. Previous work demonstrates that PERP mediates the activation of caspase-3, and we find that PERP is required for Salmonella-induced caspase-3 activation. Our combined data support a model in which SipA triggers caspase-3 activation via its cellular modulation of PERP. Since SipA can set this pathway in motion without being cleaved by caspase-3, we propose that PERP-mediated caspase-3 activation is required for the activation of SipA, and thus is a key step in the inflammatory response to S. Typhimurium infection. Our findings further our understanding of how SipA induces inflammation during S. Typhimurium infection, and also provide additional insight into how type III secreted effectors manipulate host cells.