Department of Veterinary Medicine Theses and Dissertations
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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 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 HOST-PATHOGEN INTERACTION DURING CRYPTOCOCCUS NEOFORMANS CNS INFECTION(2024) Chen, Yanli; Shi, Meiqing MS; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cryptococcus neoformans (C. neoformans) is an opportunistic fungal pathogen widely distributed in the environment globally. C. neoformans infection initiates from the lung through inhaling the spores. While most healthy individuals can clear the fungus or contain the fungus in the granuloma, immunosuppressed patients and a small group of healthy populations fail in controlling the cryptococcal fungal pulmonary infection. In those cases, C. neoformans transmigrates from the lung to the central nervous system (CNS) and causes fatal meningoencephalitis, which accounts for 112,000 deaths each year worldwide. However, we have a very limited understanding of the transmigration of C. neoformans from the bloodstream to the brain in vivo, and the mechanism involved in the clearance of the organism in the brain remains poorly understood. In this study, we first report a novel approach to quantitatively analyze the interactions between C. neoformans and brain endothelial cells in a mouse model using flow cytometry. Using this system, we show that C. neoformans was internalized by brain endothelial cells in vivo and that mice infected with acapsular or heat-killed C. neoformans yeast cells displayed a lower frequency of brain endothelial cells containing the yeast cell compared to mice infected with wild-type or viable yeast cells, respectively. We further demonstrate that brain endothelial cells were invaded by the serotype A strain (H99 strain) at a higher rate compared to the serotype D strain (52D strain). Moreover, we found that clearance of C. neoformans in the brain correlates with accumulation and pro-inflammatory M1 polarization of Ly6Chi mononuclear phagocytes and that these phagocytes play a critical role in the clearance of C. neoformans in the brain. Notably, the accumulation of Ly6Chi mononuclear phagocytes coincides with enhanced secretions of TNF and IFN-γ in the brain. TNF receptor (TNFR) signaling, but not IFN-γ receptor (IFN-γR) signaling, mediates the recruitment of Ly6Chi mononuclear phagocytes to the brain in a cell-intrinsic manner. By contrast, IFN-γ induces M1 polarization of Ly6Chi mononuclear phagocytes. Disruption of TNFR or IFN-γR signaling enhances cryptococcal growth in the brain. Thus, Ly6Chi mononuclear phagocytes act as effector cells for cryptococcal clearance in the brain, involving TNFR as well as IFN-γR signaling. Collectively, our study established that 1) internalization of C. neoformans by brain endothelial cells occurred in vivo and offered a powerful approach to quantitatively analyze fungal migration into the brain; 2) Ly6Chi mononuclear phagocytes accumulate in the brain during brain infection with C. neoformans and function as effector cells for clearance of C. neoformans in the brain involving TNFR signaling and IFN-γ signaling.Item Biological Significance of selected Ixodes scapularis Transcription Factors regulating Tick Hematophagy and Development(2023) Antara, Kazi Rifat; Pal, Utpal Professor; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lyme disease is one of the most prominent vector-borne diseases, which is transmitted by the Ixodes scapularis tick and related species, and the causative agent is the bacterial pathogen Borrelia burgdorferi. Besides I. scapularis, many other tick species are also prolific vectors of several bacterial, viral, or eukaryotic pathogens affecting humans and animals. I. scapularis possess a large genome of 2.26 Gbp, predominantly featuring repetitive DNA or transposomal elements. Although many orthologous genes are present in other arthropods and blood-borne vectors, the genome also encodes numerous unique tick-specific genes. Despite many advances in Ixodes biology and genomics, the molecular basis of their hematophagy and development remains unknown. During feeding on the host, a major tick organ like the gut undergoes remarkable yet poorly understood episodes of cell division and differentiation, accommodating a huge blood meal that can be up to 100-fold greater than their body weight. The gut, therefore, plays a vital role in blood meal acquisition, digestion, and storage, supporting the long-term survival of ticks during prolonged off-host periods of nutrient deprivation. Understanding the molecular mechanism of gut physiology, including cell division and differentiation, is an essential area of research. As transcription factors are central to the biology and development of metazoan organisms yet remain largely uncharacterized in ticks, the goal of this dissertation is to decipher the biological significance of representative groups of major development-associated transcription factors in I. scapularis that are expressed in the gut, especially during blood meal engorgement process. Among them, two of the highly upregulated transcription factors in the gut were chosen for further characterization. We show that both transcription factors, Immunoglobin-fold transcription factor (SuH) and POU domain transcription factor (Nubbin), play essential roles in tick physiology, as their knockdowns impart phenotypic defects, impacting tick feeding, development and life cycle. The latter part of the dissertation will highlight the molecular mechanism of their functions. A fundamental understanding of the molecular basis of tick biology, hematophagy, and development may contribute to developing novel strategies to curb the spread of tick-infection.Item Development of a Multilocus Sequence Typing Scheme for Avibacterium paragallinarum(2023) Harris, Alyssa Meihua; Ghanem, Mostafa; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Avibacterium paragallinarum (A. paragallinarum), the causative agent of the respiratory disease Infectious Coryza (IC) in chickens, has seen a rising incidence in the United States. Current strain differentiation is inadequate for detailed epidemiological analysis. The objective of this study was to develop a Multilocus sequence typing (MLST) scheme for A. paragallinarum for outbreak investigations and to offer a better tool for strain differentiation. By evaluating whole genome sequences and clinical samples, we designed PCR amplicons for eighteen gene segments, selected six genes for their nucleotide diversity and discrimination potential. The MLST was used to differentiate seventy-five samples. Our MLST showed greater discriminatory power than existing HPG2-based methods, aligning closely with adhoc core genome MLST in 75 tested sample. Our newly developed MLST scheme enables more accurate strain differentiation, allowing for better understanding of A. paragallinarum epidemiology and population structure to help prevention and control efforts worldwide.Item Development of improved recombinant NDV-vectored vaccines against highly pathogenic avian influenza virus (HPAIV)(2023) Roy Chowdhury, Ishita; Belov, George; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Highly pathogenic avian influenza viruses (HPAIV) are highly contagious and economically devastating poultry pathogens with a documented transmission to humans causing severe human infections with high mortality. Circulation of these viruses is of public health concern as they have the pandemic potential to mutate to increase transmissibility among humans. The diversity of zoonotic influenza viruses causing human infections is alarming and effective vaccination is needed to control these viruses. Influenza viruses particularly with H7 and H5 subtypes of HA can naturally switch to a highly pathogenic phenotype through different mechanisms. Currently available vaccines are not satisfactory as they are mostly inactivated vaccines that require labor-intensive administration methods and provide suboptimal protection of vaccinated birds. Viral vectors offer crucial advantages over traditional vaccines, including induction of outstanding antibody and cytotoxic lymphocyte responses which is important for the control of viral infections. Newcastle Disease virus (NDV) is a promising vaccine vector for HPAIV since it is highly restricted for replication in the respiratory tract of poultry, it can be easily administered, and it induces both local and systemic immune responses. H7 influenza viruses are classified into two major genetic lineages, American and Eurasian. To develop a universal anti-H7 vaccine, we generated NDV vectors expressing chimeric HA sequences covering both North American and Asian isolates. In the first project, we designed NDV-vectored vaccines against HPAI H7N8 infection. The Hemagglutinin (HA) protein of influenza viruses is responsible for virus attachment to host cell and is the major target of the humoral immune response. Accordingly, we developed vaccines against HPAIV by generating recombinant NDV vectored H7 serotype-specific vaccines expressing HA protein. We also evaluated the protective efficacy of these recombinant vaccines against highly virulent H7 challenges in both broiler chickens and turkeys and the results were promising for broiler chickens, but for turkeys the vaccination design and scheme need to be further modified. In the second part of the study, we designed some recombinant NDV-vectored vaccines with an increased level of expression of H5HA antigen. The transcriptional unit of NDV contains a major open reading frame flanked by 5’ and 3’ untranslated regions (UTRs) followed by conserved transcriptional initiation and termination control sequences. Previous studies have shown that the addition of UTRs of P, M, and F genes positively modulated foreign gene expression. Hence, we hypothesized that cognate NDV mRNA UTRs would improve the expression of a protective antigen by an NDV-vectored vaccine. We generated recombinant NDVs where the HA of the HPAIV strain H5N1 is flanked by 5’ and 3’UTRs of NDV genes and determined the growth characteristics of these recombinant viruses, their stability, the level of HA expression and their transcription and translation modulation. Both studies aimed for the advancement of NDV-vectored vaccines emphasizing the fact of better expression of the protective antigen and improved immunogenicity for avian influenza virus considering two important strains of H5 and H7.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 Immune Modulations of a Helminth Derived Protein(2023) Mekhemadhom Rajendrakumar, Arunraj; Zhu, Xiaoping XZ; Tuo, Wenbin WT; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The immune responses at the gastrointestinal mucosa modulate nematode parasite infection, initially characterized by the production of epithelium-derived, robust T helper 2 (Th2) type alarmin cytokines, such as interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP). Subsequently, the immune responses are mediated by releasing the lymphoid cell-derived Th2 immune cytokines, such as IL-13, IL-4, IL-5, IL-9, and parasite-specific antibodies. Studies have shown that parasitic nematode worms can establish a chronic infection in the intestine, even in a robust immune response. This evidence leads us to hypothesize that the nematode evolves to evade or regulate intestinal immunity through specific modulatory mechanisms that interfere with initial intestinal immune responses, allowing the nematode to survive. We used a model nematode, Heligmosomoides polygyrus bakeri (Hpb), to identify nematode-derived proteins with regulatory effects on Th2 immune cytokines during chronic infection. Through high throughput analysis, we found that a Hpb-derived protein could precisely modulate mouse immune response. The presence of the Hpb-derived protein was essential for the parasite's survival as the vaccine conferred a sterilizing immunity. As Th2 cytokines are directly associated with the pathogenesis of several inflammatory and autoimmune diseases, we are understanding how this protein regulates the function of the Th2 cytokines in vitro and in vivo and explore whether the protein could use to treat inflammatory diseases and serve as a vaccine target to control nematode infections.Item STRUCTURE-FUNCTION ANALYSES OF AN ESSENTIAL VIRULENCE DETERMINANT OF THE LYME DISEASE PATHOGEN(2022) Foor, Shelby Dimity; Pal, Utpal; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lyme Disease (LD) is a tick-borne disease caused by a group of gram-negative-like spirochetal pathogens called Borrelia burgdorferi sensu lato. The number of cases reported in the United States have dramatically increased with CDC estimating 476,000 cases annually. This multifaceted infection can spread throughout the entire body, causing clinical complications of the central nervous system, joint and heart. Early antibiotic treatment is available and effective; however, untreated patients can develop chronic symptoms, and even after antibiotics, symptoms of unknown etiology and pathogenesis can develop into post-treatment Lyme disease syndrome (PTLDS). The enzootic life cycle of B. burgdorferi is maintained typically between a small rodent and the Ixodes tick vector, where transmission occurs during tick feeding on a host. Infection establishes after B. burgdorferi is deposited in the dermis and undergoes the required shift in its protein expression profile necessary to support spirochete persistence and pathology, often highlighting protein targets for development of diagnostic, therapeutic, and preventative measures. Two such proteins identified, BB0238 and BB0323, serve as novel virulence determinants and are essential for mammalian infection. These two proteins directly interact, mutually stabilize each other post-translationally, and form an essential complex required for infection; however, their precise functions remain undetermined. In collaborative efforts, we predicted a two-domain structure of BB0238. The N-terminal domain was predicted by AI methods to harbor an antiparallel helix-turn-helix motif (HTH) followed by a third helix and a low-confidence predicted meandering segment. The C-terminal domain structure was determined by X-ray crystallography as well as predicted with high confidence to adopt an α+β fold that resembles closely that of the nuclear transport factor 2 (NTF2) superfamily. While full-length BB0238 lacks homology to singular proteins of known functions, the individual N- and C-terminal regions display structural homology to non-bacterial proteins, particularly to eukaryotic sorting, or transport proteins, suggesting that BB0238 supports an unconventional function in spirochetes. We discovered that BB0238 binds another borrelial protein annotated as BB0108, orthologs of two bacterial chaperones and foldases, the extracellular membrane anchored PrsA, and the periplasmic SurA. This identified interaction requires further investigation, however, may be important for BB0238 protein stability or assist with the novel BB0238 function discovered herein, which regulates proteolytic processing of BB0323. Furthermore, We show that key amino acid residues within the HTH stabilize BB0238 in an environment-specific manner, influence its oligomerization properties, and facilitate tick-to-mouse transmission by aiding spirochete evasion of host cellular immunity, underscoring BB0238’s ability to support microbial establishment during early mammalian infection. Together, these studies highlight the divergent evolution of multidomain spirochete proteins involved in multiplex protein-protein interactions, possibly facilitating multiple functions, which support pathogen survival and thus, represent novel targets for vaccine and therapeutic development against Lyme disease.