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All organisms produce an intracellular Zn 2+ -dependent enzyme, phosphomannose isomerase (PMI) or mannose-6 phosphate isomerase, that catalyzes the reversible conversion of mannose-6-phosphate and fructose-6-phosphate during sugar metabolism and polysaccharide biosynthesis. Unexpectedly, we discovered an additional PMI function in Borrelia burgdorferi , the pathogen of Lyme disease, where the enzyme is localized on the cell surface and binds to collagen IV-a host extracellular matrix component predominantly found in the skin. The AlphaFold 3-based structural model of B. burgdorferi PMI (BbPMI) retains the active site with tetrahedrally-coordinated Zn 2+ seen in other PMIs of known structure, residing in an elongated crevice. Ligand docking shows that the crevice can accommodate the tip trisaccharide moiety of a glycosylated asparagine residue on the collagen IV 7S domain. Low doses of a well-known PMI benzoisothiazolone inhibitor impair the growth of diverse strains of B. burgdorferi in culture, but not other tested Gram-negative or Gram-positive pathogens. Borrelia cells are even more susceptible to several other structurally related benzoisothiazolone analogs. The passive transfer of anti-BbPMI antibodies in ticks can impact spirochete transmission to mice, while the treatment of collagen IV-containing murine skin with PMI inhibitors impairs spirochete infectivity. Taken together, these results highlight a newly discovered role for BbPMI in mediating host-pathogen interactions during the spirochete infectivity process. In turn, this discovery offers an opportunity for the development of a novel therapeutic strategy to combat Lyme disease by preventing the BbPMI interaction with its host receptor, collagen IV., Importance: All organisms produce an intracellular enzyme, phosphomannose isomerase (PMI), that converts specific sugars during metabolism. Unexpectedly, we discovered an additional PMI function in Borrelia burgdorferi , the Lyme disease pathogen, where the enzyme is localized on the cell surface and binds to collagen IV-a host extracellular molecule mainly found in the skin. Low doses of PMI chemical inhibitors impair the growth of diverse strains of B. burgdorferi in culture, but not other tested bacterial pathogens. The passive transfer of anti-BbPMI antibodies in ticks can impact B. burgdorferi transmission to mice, while the treatment of collagen IV-containing murine skin with PMI inhibitors impairs infectivity. Taken together, these results highlight a newly discovered role for BbPMI in mediating host-pathogen interactions during infection. In turn, this discovery offers an opportunity for the development of a novel therapeutic strategy to combat Lyme disease by preventing BbPMI function and interaction with host collagen IV.

The bacterium responsible for Lyme disease, Borrelia burgdorferi , accumulates high levels of manganese without iron and possesses a polyploid genome, characteristics suggesting potential extreme resistance to radiation. Contrary to expectations, we report that wild-type B. burgdorferi B31 cells are radiosensitive, with a gamma-radiation survival limit for 10 6 wild-type cells of <1 kGy. Thus, we explored B. burgdorferi radiosensitivity through electron paramagnetic resonance (EPR) spectroscopy by quantitating the fraction of Mn 2+ present as antioxidant Mn 2+ metabolite complexes (H-Mn). The spirochetes displayed relatively low levels of H-Mn, in stark contrast to the extremely radiation-resistant Deinococcus radiodurans . The H-Mn content as revealed by EPR spectroscopy is sufficiently sensitive to detect small changes in radiosensitivity among B. burgdorferi strains. However, B. burgdorferi cells are significantly more sensitive than predicted by EPR, implicating their linear genome architecture as an additional explanation for radiosensitivity. We then explored the influence of the Mn 2+ -decapeptide-phosphate antioxidant complex MDP, known to shield proteins during irradiation, and showed that treatment with MDP preserves B. burgdorferi's epitopes at 5 kGy irradiation, which crucially prevents cell proliferation. This finding defines some of the pivotal mechanisms that B. burgdorferi evolved to survive oxidative conditions experienced with tick and mammal immune responses. These observations also provide an opportunity for innovative vaccine development strategies employing ionizing radiation to disrupt the B. burgdorferi genome, while maintaining antigenic potency. These fresh insights extend our understanding of the unique biology of B. burgdorferi and open new avenues for considering novel whole-cell Lyme disease vaccines using MDP and irradiation-based inactivation.IMPORTANCEThe study highlights that electron paramagnetic resonance (EPR) spectroscopy is sufficiently sensitive to detect small differences in radiation resistance among Borrelia burgdorferi strains based on their population of Mn 2+ -metabolite complexes (H-Mn). B. burgdorferi appears to have evolved a system not to protect from irradiation, but presumably to protect from oxidative stress when cyclically transmitted from tick to mammalian host and back. These data also suggest a path forward in the development of novel vaccines against spirochete infections, including Lyme disease, through preparation involving the synthetic Mn 2+ -decapeptide-phosphate antioxidant complex MDP to provide B. burgdorferi epitope protection during sterilizing gamma-irradiation that eliminates growth. Given the current lack of effective whole-cell vaccines for Lyme disease, this research identifies a potential strategy for developing alternative radiation-inactivated, yet highly effective vaccines., Competing Interests: The authors declare no conflict of interest.

Borrelia burgdorferi , the pathogen of Lyme disease, differentially produces many outer surface proteins (Osp), some of which represent the most abundant membrane proteins, such as OspA, OspB, and OspC. In cultured bacteria, these proteins can account for a substantial fraction of the total cellular or membrane proteins, posing challenges to the identification and analysis of non-abundant proteins, which could serve as novel pathogen detection markers or as vaccine candidates. Herein, we introduced serial mutations to remove these abundant Osps and generated a B. burgdorferi mutant deficient in OspA, OspB, and OspC in an infectious 297-isolate background, designated as Osp ABC - mutant. Compared to parental isolate, the mutant did not reflect growth defects in the cultured medium but showed differential mRNA expression of representative tested genes, in addition to gross changes in cellular and membrane protein profiles. The analysis of differentially detectable protein contents of the Osp ABC - mutant, as compared to the wild type, by two-dimensional gel electrophoresis followed by liquid chromatography-mass spectrometry, identified several spirochete proteins that are dominated by proteins of unknown functions, as well as membrane transporters, chaperons, and metabolic enzymes. We produced recombinant forms of two of these represented proteins, BBA34 and BB0238, and showed that these proteins are detectable during spirochete infection in the tick-borne murine model of Lyme borreliosis and thus serve as potential antigenic markers of the infection.IMPORTANCEThe present manuscript employed a systemic approach to identify non-abundant proteins in cultured Borrelia burgdorferi that are otherwise masked or hidden due to the overwhelming presence of abundant Osps like OspA, OspB, and OspC. As these Osps are either absent or transiently expressed in mammals, we performed a proof-of-concept study in which their removal allowed the analysis of otherwise less abundant antigens in OspABC-deficient mutants and identified several immunogenic proteins, including BBA34 and BB0238. These antigens could serve as novel vaccine candidates and/or genetic markers of Lyme borreliosis, promoting new research in the clinical diagnosis and prevention of Lyme disease., Competing Interests: The authors declare no conflict of interest.

Importance: Lyme disease is a major tick-borne infection caused by a bacterial pathogen called Borrelia burgdorferi , which is transmitted by ticks and affects hundreds of thousands of people every year. These bacterial pathogens are distinct from other genera of microbes because of their distinct features and ability to transmit a multi-system infection to a range of vertebrates, including humans. Progress in understanding the infection biology of Lyme disease, and thus advancements towards its prevention, are hindered by an incomplete understanding of the microbiology of B. burgdorferi , partly due to the occurrence of many unique borrelial proteins that are structurally unrelated to proteins of known functions yet are indispensable for pathogen survival. We herein report the use of diverse technologies to examine the structure and function of a unique B. burgdorferi protein, annotated as BB0238-an essential virulence determinant. We show that the protein is structurally organized into two distinct domains, is involved in multiplex protein-protein interactions, and facilitates tick-to-mouse pathogen transmission by aiding microbial evasion of early host cellular immunity. We believe that our findings will further enrich our understanding of the microbiology of B. burgdorferi, potentially impacting the future development of novel prevention strategies against a widespread tick-transmitted infection., Competing Interests: The authors declare no conflict of interest.

The Borrelia burgdorferi BB0323 protein undergoes a complex yet poorly defined proteolytic maturation event that generates N-terminal and C-terminal proteins with essential functions in cell growth and infection. Here, we report that a borrelial protease, B. burgdorferi high temperature requirement A protease (BbHtrA), cleaves BB0323 between asparagine (N) and leucine (L) at positions 236 and 237, while the replacement of these residues with alanine in the mutant protein prevents its cleavage, despite preserving its normal secondary structure. The N-terminal BB0323 protein binds BbHtrA, but its cleavage site mutant displays deficiency in such interaction. An isogenic borrelial mutant with NL-to-AA substitution in BB0323 (referred to as Bb bb0323NL ) maintains normal growth yet is impaired for infection of mice or transmission from infected ticks. Notably, the BB0323 protein is still processed in Bb bb0323NL , albeit with lower levels of mature N-terminal BB0323 protein and multiple aberrantly processed polypeptides, which could result from nonspecific cleavages at other asparagine and leucine residues in the protein. The lack of infectivity of Bb bb0323NL is likely due to the impaired abundance or stoichiometry of a protein complex involving BB0238, another spirochete protein. Together, these studies highlight that a precise proteolytic event and a particular protein-protein interaction, involving multiple borrelial virulence determinants, are mutually inclusive and interconnected, playing essential roles in the infectivity of Lyme disease pathogens.