College of Agriculture & Natural Resources
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Item ZIKA VIRUS RECRUITS CELLULAR PROTEINS TO SUPPORT ITS REPLICATION(2024) Chang, Peixi; Zhang, Yanjin YJ; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Zika virus (ZIKV) is a mosquito-borne pathogen with a massive impact on global public health due to its association with severe neurological complications, including microcephaly in newborns and Guillain-Barré syndrome in adults. The ZIKV epidemic in the Americas in 2015-2016 and its continuing spread in tropical regions have highlighted the urgent need to understand the molecular mechanisms of viral replication to develop effective antiviral strategies. However, many aspects of how ZIKV interacts with host cells remain unclear. This study identifies and characterizes host factors contributing to ZIKV replication. First, karyopherin alpha 6 (KPNA6) contributes to ZIKV replication by interacting with the ZIKV non-structural protein NS2B. Characterization and mutational analyses identified two essential amino acid residues within NS2B that are critical for interacting with KPNA6. The substitution of these two residues of NS2B in an infectious ZIKV cDNA clone resulted in a significant reduction in viral replication, suggesting that the NS2B-KPNA6 interaction plays a vital role in the viral life cycle. Further studies found that KPNA6 contributes to ZIKV RNA synthesis. Mass spectrometry analysis of the KPNA6 interactome showed that KPNA6 interacts with proteins involved in RNA synthesis, suggesting that ZIKV recruits these factors by promoting KPNA6-binding. Second, this study developed an effective method to isolate the ZIKV replication complex, a membranous structure where viral RNA is synthesized. Proteomic analysis of the isolated complex led to identifying numerous host proteins associated with the viral replication machinery. Among these proteins, human replication factor C subunit 2 (RFC2), an accessory factor involved in DNA replication and repair, was discovered to facilitate ZIKV replication, making it a potential target for therapeutic interventions. In conclusion, this study reveals crucial host factors essential for ZIKV infection and replication and provides insights into the ZIKV-cell interactions. These findings offer new possibilities for developing novel antiviral strategies for controlling future viral outbreaks.Item ROLE OF TRPV4 MECHANOSENSING REGULATING MACROPHAGE FUNCTIONS IN INFLAMMATORY DISEASES(2024) Dutta, Bidisha; Rahaman, Shaik O; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Macrophages are the most versatile cells of the hematopoietic system with roles in homeostasis, host-tissue development, innate immune response and tissue repair. Although the inflammatory activation and maintenance signals are tightly regulated, an imbalance in them results in unchecked inflammation resulting in cellular and tissue damage. Macrophages can affect most if not all phases of inflammation owing to their ability to adopt distinct functional states, secrete cytokines and phagocytose pathogens and debris. Recent evidence suggests that beyond biochemical cues, mechanical forces, like changing matrix stiffness in the tissue microenvironment, can shape immune cell functions involved in inflammation. These cells convert mechanical stimuli to biochemical signals in a process called mechanotransduction, regulating a multitude of cellular functions. However, knowledge about the molecular mediators of mechanotransduction and their functions in macrophage phenotypic and functional change is largely missing, highlighting the need for studying mechanosensory molecules such as ion channels. The present study focuses on the role of a specific mechanosensitive ion channel, Transient Receptor Potential Vanilloid 4 (TRPV4), in the regulation of macrophage mediated inflammatory responses. Given its emerging role in inflammatory diseases like fibrosis, arthritis, foreign body response (FBR), TRPV4’s contribution to macrophage behavior in inflammation is of growing interest. Employing cellular imaging and molecular biology techniques such as Ca2+ influx assays, immunohistochemistry, immunoblotting, and single nuclei RNA sequencing we delineate mechanisms by which biomechanical stimuli-mediated activation of TRPV4 affects macrophage function. We elucidate TRPV4’s role in macrophage mechanotransduction, providing a mechanistic understanding of inflammatory disease pathophysiology which could lead to the development of potential therapeutics for disease intervention.Item MEDIATION OF CORTICOSTERONE-INDUCED GROWTH HORMONE GENE EXPRESSION IN CHICKEN EMBRYONIC PITUITARY CELLS: IDENTIFICATION OF TRANS-ACTING FACTORS AND A NOVEL PITUITARY CELL TYPE(2024) Liu, Kuan Ling; Porter, Tom E.; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Growth hormone (GH) is responsible for up to 30% of growth in broiler chickens. Somatotrophs, or GH secreting cells, begin to differentiate around embryonic day (e)14, in conjunction with an increase in the primary plasma glucocorticoid (GC) corticosterone (CORT). CORT treatment of e11 chicken embryonic pituitary (CEP) cells induces premature GH secretion. This GC-induced process involves trans-acting factors because the GH gene lacks a canonical GC response element (GRE). In addition to the binding of ETS1 and the GC receptor (GR) to the GC-responsive region (GCRR; 1045/ 964), we hypothesize that there are other regulatory factors necessary for CORT responsiveness. By modifying the pGL3_-1742/+25 GH-luciferase reporter, we have constructed various other GH-luciferase reporters and analyzed them for promoter activity in response to CORT treatment. We identified a putative distal (d) ETS-Like 1 (ELK1) binding site that is necessary. The proximal (p)PIT1 site and pTATA box were also identified to be critical for CORT induction of the GH gene. Interestingly, cloning multiple copies of the extended GCRR (eGCRR; -1067/-900) further increased promoter activity in an additive manner under both basal and CORT treated conditions. Through single-cell RNA sequencing (scRNAseq), 8 members of the ETS family of transcription factors were identified in > 5% of the somatotroph population. Commercial antibodies were validated, and human (h)ETV1, hELF2, hELK3, and hETV6 antibodies were confirmed to recognize their recombinant chicken ortholog and to identify their corresponding protein in e11 CEP cells. Results from chromatin immunoprecipitation quantitative PCR suggest that multiple ETS members are involved in CORT induction of the GH gene with more evidence pointing towards ELF2 and ELK3. Identifying trans-acting factors for the GH gene inducible by CORT allows for better understanding of endogenous GH regulation in chickens. Further analysis of the scRNAseq data from e11 CEP cells revealed a cluster of cells expressing genes for more than one hormone-producing cell type (“premature nebulous” cluster). Within the premature nebulous cluster, a large population (~30%) was co-expressing proopiomelanocortin (POMC) and growth hormone (GH). We named this novel cell population the cortico-somatotrophs. Through RNA fluorescent in-situ hybridization (RNA-FISH) and dual label immunofluorescence, we verified the existence of the cortico-somatotrophs at both the mRNA and protein level, respectively. Cortico-somatotrophs were also shown to share genes for receptors normally specific to both corticotrophs (CRH-R1) and somatotrophs (GHRHR). Additionally, in response to CORT treatment, the cortico somatotrophs showed an increase in GH as well as a decrease in POMC mRNA levels. The discovery of the cortico-somatotrophs suggests a modification to the current dogma on pituitary cell lineages, where corticotrophs and somatotrophs may have overlapping developmental pathways. In conclusion, our discovery of the cortico somatotrophs has furthered our understanding of CEP development and opened the door for further exploration of the cell lineages during pituitary development.Item Characterization of the GBF1-Arf1 axis in enterovirus RNA replication(2024) Gabaglio Velazquez, Samuel Maria; Belov, George; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Enterovirus genus includes many known and emerging pathogens, such as poliovirus, enteroviruses A71 and D68, rhinoviruses, and others. Enterovirus infection induces the massive remodeling of intracellular membranes and the development of specialized domains harboring viral replication complexes, called replication organelles. The cellular protein Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) is essential for the replication of enteroviruses, but its molecular role in the replication process is unclear. In uninfected cells, GBF1 activates small GTPases of the Arf family and coordinates multiple steps of membrane metabolism, including the functioning of the cellular secretory pathway. The nonstructural protein 3A of poliovirus and other enteroviruses directly interact with and recruits GBF1 to the replication organelles. Moreover, enterovirus infection induces the massive recruitment of all isoforms of the small cellular Arf GTPases to the replication organelles, but the mechanistic role of these proteins in the replication process is not understood either. Here, we sought to characterize the role of the GBF1-Arf1 axis in enterovirus replication. First, we systematically investigated the conserved elements of GBF1 to understand which determinants are important to support poliovirus replication. We demonstrated that multiple GBF1 mutants inactive in cellular metabolism could still be fully functional in the replication complexes. Our results showed that the Arf-activating property, but not the primary structure of the Sec7 catalytic domain is essential for viral replication. They also suggest a redundant mechanism for recruiting GBF1 to the replication sites. This mechanism depends not only on the direct interaction of the protein with the viral protein 3A but also on elements located in the noncatalytic C-terminal domains of GBF1. Next, we investigated the distribution of viral proteins and Arf1 on the replication organelles and their biochemical environment. Pulse-labeling of viral RNA with 5-ethynyl uridine showed that active RNA replication is associated with Arf1-enriched membranes. We observed that Arf1 forms isolated microdomains in the replication organelles and that viral antigens are localized in both Arf1-depleted and Arf1-enriched microdomains. We investigated the viral protein composition of the Arf1-enriched membranes using peroxidase-based proximity biotinylation. Viral protein biotinylation was detected as early as 3 h.p.i., and the non-cleaved fragments of the viral polyprotein were overrepresented in the Arf1-enriched domains. Furthermore, we show that after 4 h.p.i. viral proteins could be efficiently biotinylated only upon digitonin permeabilization of the replication organelle membranes, while such permeabilization inhibited the Arf1 biotinylation signal at the Golgi in non-infected cells. Together, these data support a model that recruitment of GBF1 to the replication organelles generates foci of activated Arfs on the membranes, which further differentiate into specific microdomains through the recruitment of a specific complex of viral proteins and cellular Arf effectors likely needed to establish the lipid and protein composition required for viral replication.Item Three Clostridium species with Health Imparting Properties: In vitro Screening for Probiotic Potential(2024) Mochama, Victor Moronge; Obanda, Diana; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This research aimed to unlock the probiotic potential of the genus Clostridium, which is often overshadowed by the predominant focus on pathogenic species. The study specifically targeted three promising Clostridium species: C. disporicum, C. celatum, and C. vincentii, which have shown potential in mitigating diet-induced obesity. Despite the challenges presented by the anaerobic growth requirements of Clostridium bacteria, the study capitalized on their capacity to sporulate. This characteristic provides an avenue to use them as probiotics, with resilient and dormant spores capable of surviving food processing and harsh stomach conditions. The resilience of these spores was examined by exposing them to oxygen, heat, gastrointestinal juices, and bile salts. The spores survived oxygen exposure, exhibited resilience to both bile salts and gastric acids, and demonstrated a survival temperature of 70°C. When exposed to suitable germination conditions in vitro, the spores successfully germinated. The study assessed the colonization potential of the bacteria by evaluating their adhesion ability, and all bacteria were found to have the adhesion ability. Furthermore, a safety assessment was conducted by examining hemolytic activity and antibiotic susceptibility to selected antibiotics. The bacteria were found to be susceptible to the antibiotics and did not exhibit hemolytic activity. Bile salt hydrolase (BSH) activity and antibacterial activities were also assessed, and none of the bacteria exhibited BSH activity or antibacterial activity. Antioxidant tests revealed that C. vincentii had the highest antioxidant properties. Assessment of anti-inflammatory properties showed that C. celatum downregulated the gene expression of cytokine inflammation markers IL-6, IL-1, and iNOS while upregulating TGF-β expression. In summary all 3 bacterial species showed good probiotic potential from the in vitro tests. Particularly the formation of resistant spores that later germinated to vegetative cells that produced molecular patterns with antioxidant and anti-inflammatory properties. This necessitates further studies on their probiotic properties.Item COORDINATED TRAFFICKING OF HEME TRANSPORTERS BY CARGO SORTING COMPLEXES IS ESSENTIAL FOR ORGANISMAL HEME HOMEOSTASIS(2025) Dutt, Sohini; Hamza, Iqbal IH; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme, an iron-containing organic ring, is a vital cofactor responsible for diverse biologicalfunctions and is the major source of bioavailable iron in the human diet. As a hydrophobic and cytotoxic cofactor, heme must be transported in a highly controlled manner through membranes via specific intra- and inter-cellular pathways. However, the genes and pathways responsible for heme trafficking remain poorly understood. Unlike other metazoans, Caenorhabditis elegans cannot synthesize heme but requires heme for sustenance. Thus, C. elegans is an ideal animal model to identify heme trafficking pathways as it permits organismal heme homeostasis to be directly manipulated by controlling environmental heme. Heme is imported apically into the intestine by HRG-1-related permeases and exported basolaterally by MRP-5/ABCC5 to extra- intestinal tissues. Loss of mrp-5 causes embryonic lethality that can be suppressed by dietary heme supplementation raising the possibility that MRP-5-independent heme export pathways must exist. Here we show, by performing a forward genetic screen in mrp-5 null mutants, that loss of the vesicular cargo sorting Adaptor Protein complexes (AP-3) fully rescues mrp-5 lethality and restores heme homeostasis. Remarkably, intestinal heme accumulation due to mrp-5-deficiency causes a concomitant deficit in the lysosomal heme importer HRG-1 abundance and localization. Loss of both MRP-5 and AP-3 subunits resurrects HRG-1 levels and localization, thus underscoring the crucial role of HRG-1 in dictating mrp-5 mutant phenotypes. In the absence of MRP-5, heme is exported by SLC49A3 homolog, a previously uncharacterized transporter. Live- cell imaging reveals vesicular coalescence that facilitates heme transfer between the importers and exporters at the interface of lysosomal-related organelle. These results define a mechanistic model for metazoan heme trafficking and identifies SLC49A3 as a promising candidate for heme export in mammals.Item EVALUATION OF THE COPPER HOMEOSTASIS AND SILVER RESISTANCE ISLAND AND ITS ROLE IN PERSISTENCE OF SALMONELLA ENTERICA(2023) Haendiges, Julie; Tikekar, Rohan; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Salmonella enterica is one of the leading bacterial cause of foodborne illness in the UnitedStates. Although there are many serovars, only a small subset causes human illness. Since Salmonella is ubiquitous in the environment, the Food and Drug Administration has established regulations for food processors to ensure that the products are free of contamination. Low-moisture foods are commonly ready-to-eat, and due to the low water activity do not promote growth of bacteria. However, Salmonella has been shown to persist in these foods. There havebeen two outbreaks and multiple recalls in the United States due to contaminated pistachios. Based on a retrospective study, results show that there is evidence of a contamination in the growing orchard and a significant number of Salmonella isolates from the environments contain the Copper Homeostasis and Silver Resistance Island (CHASRI) cassette. This raises several questions: what is the prevalence of CHASRI among different Salmonella isolates from food and environmental sources? Does presence of CHASRI enable Salmonella to survive better against copper stress? And whether presence of CHASRI provide cross-protection against other stresses such as desiccation and thermal treatment? This dissertation attempts to answer those questions. The prevalence of the CHASRI in Salmonella was determined by the use of publicly availablewhole genome sequencing data. The CHASRI was found in 61 different serovars and types of sources. The presence of the CHASRI in isolates from low-moisture foods that have caused previous outbreaks (peanut butter, nuts, spices) was interesting to note, and leads to future studies on correlations between this island and virulence. Based on results of phylogenetic analysis of CHASRI sequences from closed genomes, we determined there were four types of CHASRI found in Salmonella. Traditionally, the Salmonella Genomic Island-4 (SGI-4) is found but in addition the CHASRI can incorporate by itself, within a variant of SGI-4, or via a rare plasmid. Interestingly, the sequence of the CHASRI from SGI-4 and the variant SGI-4 were highly different. The high SNP differences in sequence along with the difference in the arsenic operon led to the conclusion that these variants arose independently. A Salmonella Senftenberg strain (CFSAN047523), isolated from pistachios, was used to createthree knockouts (∆cus, ∆pco, and ∆CHASRI). Previous studies have looked at the minimum inhibitory concentration (MIC) of strains with and without the CHASRI but have omitted the minimum bactericidal concentration (MBC). In this study, we used the knockouts to test for both MIC and MBC. While the MIC was similar for the strains, the MBC was greater in the wild type and partial CHASRI knockouts. Growth and inactivation kinetics were measured in different concentrations of copper sulfate. At higher levels of copper sulfate, the presence of the CHASRI made cells more resilient to inactivation by copper sulfate. Evidence shows that the stress response in Salmonella has the ability to crosstalk and provideprotection against multiple stresses. To investigate this phenomenon further, our isolates were tested against a multitude of stresses to evaluate for cross-protection that may be due to theCHASRI. Cells undergoing copper stress were better equipped to survive lethal copper concentrations and desiccation if the CHASRI was present. The presence of Salmonella in final pistachio products that have been fully processed identifies that some adaptation and stress response is occurring in the processing facility. Inoculated pistachios with the wild type and ∆CHASRI strain were thermally processed to test for survivors. This study showed that the presence of the CHASRI gave the isolate an advantage to survive thermal processing after desiccation. Overall, this study presents the prevalence of the CHASRI in Salmonella enterica as well as theimportant role it plays in copper tolerance. The evidence of cross-protection and tolerance to copper leads to future research regarding gene expression and virulence assessment.Item MOLECULAR DISSECTION OF BORRELIA BURGDORFERI BB0323 PROTEIN COMPLEX SUPPORTING MICROBIAL BIOLOGY, INFECTIVITY, AND AS A NOVEL THERAPEUTIC TARGET(2023) Bista, Sandhya; Pal, Utpal Dr.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lyme disease (LD), also known as Lyme borreliosis, is the most common vector-borne disease in the United States, caused by the gram-negative bacteria of the Borrelia burgdorferi sensu lato group. This atypical bacterial group features distinct genomic and antigenic elements, does not possess any classical toxins, and the pathogenesis of LD is primarily due to the immune activity of the host. These multi-organotrophic spirochetes can elicit severe clinical complications in susceptible hosts, including neuroborreliosis, carditis, and arthritis. If diagnosed early, the disease can be treated with a conventional antibiotic regimen; however, persistent, or relapsing symptoms later develop in a subset of patients. Six months to a year after the antibiotic treatment, up to 20% of the patients can experience various subjective symptoms pertaining to pain, cognitive dysfunction, or other neurological complications, collectively termed Post Treatment Lyme Disease Syndrome (PTLDS). The diagnosis, etiology, and treatment of PTLDS remain currently unknown. To better understand microbial pathogenesis, we have characterized a select set of structurally unique spirochete gene products that act as novel virulence determinants and support microbial infection in mammals. The current study focused on the BB0323 protein of B. burgdorferi, a unique and multifunctional virulence determinant undergoing a complex post-translational maturation process. The maturation, stability, and functions of BB0323 require multifaceted protein-protein interaction (PPI) events involving specific B. burgdorferi proteins, such as a protease-chaperone called BbHtrA, and a membrane-associated protein of unknown function annotated as BB0238. In our current study, we have further dissected the biological significances of the protein-protein interaction complex (PPI), either involving BbHtrA: BB0323 and BB0323:BB0238. The latter PPI event was more thoroughly investigated for its role in spirochete biology and infection and as a novel target for therapeutic intervention against B. burgdorferi infection. We identified a cleavage site where BB0323 full-length protein cleaves into N and C termini by BbHtrA. Subsequently, we have introduced point mutations in the recombinant BB0323 (at the cleavage site for BbHtrA- NL residues replaced with AA), as well as generated an isogenic B. burgdorferi isolates (Bbbb0323NL) with the point mutations in native BB0323. Further analyses show that the cleavage site mutated BB0323 protein could not be processed by the recombinant BbHtrA. Notably, despite the inability of BbHtrA to process BB0323 in vitro, within Bbbb0323NL, BB0323 could indeed be processed to some degree, which yields a basal level of mature N-terminal protein. Notably, in these B. burgdorferi cells, at least two other BB0323 polypeptides of lower molecular weight (less than 27 kDa of mature N-term BB0323) were also produced, possibly due to the action of BbHtrA on non-specific sites. However, the Bbbb0323NL mutants were non-infectious in the murine host, demonstrating the importance of precise cleavage of BB0323 full-length protein and optimal production of N-terminal, which needed to form a complex with another PPI partner, BB0238. Overall, these results further underscored the event of BbHtrA and BB0323 interaction for processing the latter protein as an essential prerequisite for spirochete infection in mammals. Our previous studies have shown that BB0323 N-terminal and BB0238 interact and post-translationally stabilize each other. We used an interaction-deficient borrelial mutant, replacing the BB0323 interaction motif in BB238 (termed as bb0238 Delta Interaction Motif, or bb0238∆IM), which despite showing no growth defects in vitro or other abnormalities, is unable to infect mammalian host. We, therefore, explored the possibility of using the BB0323:BB0238 complex as a novel therapeutic target to combat B. burgdorferi infection in mammals. We first examined whether bb0238∆IM mutants (without interaction motifs) can persist in mice for a long term or could be acquired by naïve ticks. The results show that, unlike the wild type or another B. burgdorferi mutant, The bb0238∆IM could not establish the infection in mice and, as a result, could not be acquired by the ticks, suggesting blockade of BB0323:BB0238 interaction by small molecules could be a novel therapeutic approach to combat incidence of LD. An AlphaLisa assay platform was developed in our lab to monitor BB0323-BB0238 PPI on a high-throughput basis using 384-well microtiter plates, which was then miniaturized to 1536 well at the National Center for Advancing Translational Sciences (NCATS) in a collaborative effort. An AlphaLisa quantitative HTS later screened several small molecule libraries available at NCATS, which were further filtered by counter assays, and a selected set of 84 compounds was tested in a secondary, cell-based assay for cell-permeable compounds that impair BB0323-BB0238 interaction with spirochete cells. A B. burgdorferi cell-based assay comprising a dot-blot assay and regrowth assay was developed to examine the PPI inhibitory activities of the molecules inside the cells. We finally selected one of the compounds, Lomibuvir, for the in vivo studies and demonstrated its PPI inhibitory activity in an in vitro experiment. A pharmacokinetic study in mice showed an increase in the level of the compound in plasma and liver over 21 days. Additional in vivo efficacy studies of Lomibuvir to reduce B. burgdorferi infection in mice were performed using vehicle and ceftriaxone as negative and positive controls, respectively. The results showed that the bacterial load in the skin and heart of the mice was significantly lower in the Lomibuvir-treated group, as compared to the vehicle-treated animals; however, the effect was not as dramatically effective as the antibiotic (ceftriaxone) treatment groups. While future medicinal chemistry approaches could be adopted to further enhance the impact of Lomibuvir as an anti-B. burgdorferi agent, to the best of knowledge, is the first proof-of-concept study that highlights the utility of targeting borrelial PPI events as a possible therapeutic target of Lyme disease.Item A NOVEL IXODES SCAPULARIS PROTEIN DICTATES TICK HEMATOPHAGY AND CUTICLE INTEGRITY, IMPACTING TICK DEVELOPMENT(2023) DUTTA, SHRABONI; Pal, Utpal Dr.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ticks are prevalent throughout the world and are capable of transmitting a variety of pathogens (e.g., bacteria, protozoa, and viruses) to humans. Incidence rates for tick-borne diseases (TBD) are also increasing globally, and effective vaccinations to combat tick infestations and TBD transmission remain a critical unmet need. Of the six major tick genera that spread human illnesses worldwide, Ixodes ticks are the most prevalent. Specifically, Ixodes scapularis (also known as the blacklegged or deer tick) is an obligate blood-feeding arthropod that transmits several human and animal pathogens that include Borrelia burgdorferi sensu lato complex – the causative agent for Lyme disease. Unlike many hematophagous insects and soft ticks, I. scapularis (hard ticks) remain attached to their hosts for several days and are capable of uptaking bloodmeals that are 100 times greater than their initial body weight. A large and nutrient-dense bloodmeal is essential for their sub-adult and adult development processes and fecundity. However, the molecular and cellular processes that regulate tick blood feeding (hematophagy) and development have not been extensively elucidated. Therefore, our major objective is to characterize tick molecular components that are critical in the tick parasitism and life cycle in order to develop new strategies to combat tick infestations and spread of tick-borne diseases. Herein, we describe the structural and functional properties of a newly identified I. scapularis protein isolated from the partially fed nymphal ticks. Although the protein displays minor homology to proteins of known functions, structurally, it resembles some features of arthropod Odorant Binding Proteins (OBP). Therefore, we refer to this protein as, Ixodes Gut OBP (IGOBP). We show that the knockdown of IGOBP via RNA interference in ticks results in impaired blood feeding (hematophagy) and significantly decreases their post-fed weights. In addition, systemic IGOBP knockdown gives rise to aberrant phenotypes, significantly reduces tick molting rate, and compromises the structural integrity of the cuticle, specifically the flexible alloscutum components. Notably, IGOBP knockdown has profound effects on the molting efficacy and fitness of females than males. This is likely due to the fact that female adults consume a greater volume of bloodmeal than male adults, necessitating a more pronounced expansion of the alloscutum. Subsequently, our RNA sequencing data identifies multiple genes whose expressions are regulated by IGOBP. The underlying mechanism of possible IGOBP or associated gene functions may aid in identifying future targets for anti-tick vaccines. In summary, our studies characterized a novel I. scapularis protein revealing that the protein is essential for tick hematophagy and development. To the best of our knowledge, this is the first characterization of a tick odorant-binding protein (OBP), using structural and functional genomic tools that unearthed the unique and possibly multifunctional role of IGOBP in vector biology and parasitism. We anticipate that the presented data will enhance our fundamental understanding of tick biology and contribute to the development of potential anti-tick measures.Item ANTAGONISTIC MECHANISM OF METABOLITES FROM LACTOBACILLUS CASEI AGAINST FOODBORNE ENTEROHEMORRHAGIC ESCHERICHIA COLI(2022) Aditya, Arpita; Biswas, Debabrata; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Enterohemorrhagic Escherichia coli O157: H7 (EHEC), a foodborne enteropathogen, remains a significant public health concern since its discovery in 1982. With an incredibly low infectious dose (10-100 bacteria), this pathogen can cause self-limiting diarrhea, vomiting, and abdominal cramps. However, more complicated disease conditions such as bloody diarrhea or hemolytic colitis have been known to develop depending on the serotype involved in the infection, and on immune status and/or age of the patients. Due to its Shiga toxin (Stx) production ability, EHEC infection may lead to a kidney-related problem known as hemolytic uremic syndrome (HUS), which requires advanced medical care. Unlike other bacterial illnesses, therapeutic administration of antibiotics to treat EHEC infections is not recommended due to their controversial association with Stx production. As a result, only preventative/prophylactic and immune-supportive strategies are followed for EHEC infections. Using the antibacterial properties of probiotic bacteria and the metabolites they produce are promising alternative strategies for preventing EHEC infections. We have targeted the probiotic bacteria Lactobacillus casei to determine the mechanism of this alternative strategy. In our study, we have executed microbiological, molecular, chromatographic, and metagenomic approaches to determine the antagonistic mechanisms of action of their metabolites, specifically conjugated linoleic acid (CLA) produced by Lactobacillus casei, against the growth and metabolism of EHEC. The metabolites of wild-type L. casei (LCwt) were augmented by supplementing it with a prebiotic-like dietary component, namely peanut flour (PF) (LCwt+PF), while another LCwt was also genetically engineered (LCCLA) to over convert CLA from linoleic acid (LA). These modifications showed effective results in controlling EHEC both in vitro and in ex vivo conditions. Total metabolites present in cell-free culture supernatant (CFCS) of LCwt, LCwt+PF, and LCCLA were able to control the growth of EHEC without negatively hampering the relative abundance of Firmicutes and Bacteroidetes present in rumen fluid (RF). Among these CFCSs, CFCSCLA exerted the most desirable outcome by eliminating EHEC. In vitro studies demonstrated that, a lower concentration of purified CLA worked synergistically with other metabolites of LCwt and augmented their inhibitory activity against EHEC. The orchestrated effect of metabolites has been observed to downregulate the virulence genes, disrupt the cell membrane, interfere with cell division, and damage their genomic DNA. The probable effect of these metabolites, specifically CLA, on Stx production and neutralization was also investigated by assessing host cell cytotoxicity. Total metabolites of Lactobacillus spp. as well as CLA itself, showed improvement in cell survivability when exposed to Stx. Our findings established a ground to explore the effect of specific metabolites obtained from probiotic bacteria in control and prevention of EHEC. The findings also showed a promising association of purified CLA in neutralizing Stx which can be further explored to use it in therapeutic purposes.