Antibody-blocking of a tick transporter impairs Anaplasma phagocytophilum colonization in Haemaphysalis longicornis ticks.

The invasive Asian longhorned tick Haemaphysalis longicornis that vectors and transmits several animal pathogens is significantly expanding in the United States. Recent studies report that these ticks also harbor human pathogens including Borrelia burgdorferi sensu lato, Babesia microti, and Anaplasma phagocytophilum. Therefore, studies that address the interactions of these ticks with human pathogens are important. In this study, we report the characterization of H. longicornis organic anion-transporting polypeptides (OATPs) in interactions of these ticks with A. phagocytophilum. Using OATP-signature sequence, we identified six OATPs in the H. longicornis genome. Bioinformatic analysis revealed that H. longicornis OATPs are closer to other tick orthologs rather than to mammalian counterparts. Quantitative real-time PCR analysis revealed that OATPs are highly expressed in immature stages when compared to mature stages of these ticks. In addition, we noted that the presence of A. phagocytophilum upregulates a specific OATP in these ticks. We also noted that exogenous treatment of H. longicornis with xanthurenic acid, a tryptophan metabolite, influenced OATP expression in these ticks. Immunoblotting analysis revealed that antibody generated against Ixodes scapularis OATP cross-reacted with H. longicornis OATP. Furthermore, treatment of H. longicornis with OATP antibody impaired colonization of A. phagocytophilum in these ticks. These results not only provide evidence that the OATP-tryptophan pathway is important for A. phagocytophilum survival in H. longicornis ticks but also indicate OATP as a promising candidate for the development of a universal anti-tick vaccine to target this bacterium and perhaps other rickettsial pathogens of medical importance.

Members of the paralogous gene family 12 from the Lyme disease agent Borrelia burgdorferi are non-specific DNA-binding proteins.

Lyme disease is the most prevalent vector-borne infectious disease in Europe and the USA. Borrelia burgdorferi, as the causative agent of Lyme disease, is transmitted to the mammalian host during the tick blood meal. To adapt to the different encountered environments, Borrelia has adjusted the expression pattern of various, mostly outer surface proteins. The function of most B. burgdorferi outer surface proteins remains unknown. We determined the crystal structure of a previously uncharacterized B. burgdorferi outer surface protein BBK01, known to belong to the paralogous gene family 12 (PFam12) as one of its five members. PFam12 members are shown to be upregulated as the tick starts its blood meal. Structural analysis of BBK01 revealed similarity to the coiled coil domain of structural maintenance of chromosomes (SMC) protein family members, while functional studies indicated that all PFam12 members are non-specific DNA-binding proteins. The residues involved in DNA binding were identified and probed by site-directed mutagenesis. The combination of SMC-like proteins being attached to the outer membrane and exposed to the environment or located in the periplasm, as observed in the case of PFam12 members, and displaying the ability to bind DNA, represents a unique feature previously not observed in bacteria.

Borrelia burgdorferi PlzA is a cyclic-di-GMP dependent DNA and RNA binding protein.

The PilZ domain-containing protein, PlzA, is the only known cyclic di-GMP binding protein encoded by all Lyme disease spirochetes. PlzA has been implicated in the regulation of many borrelial processes, but the effector mechanism of PlzA was not previously known. Here, we report that PlzA can bind DNA and RNA and that nucleic acid binding requires c-di-GMP, with the affinity of PlzA for nucleic acids increasing as concentrations of c-di-GMP were increased. A mutant PlzA that is incapable of binding c-di-GMP did not bind to any tested nucleic acids. We also determined that PlzA interacts predominantly with the major groove of DNA and that sequence length and G-C content play a role in DNA binding affinity. PlzA is a dual-domain protein with a PilZ-like N-terminal domain linked to a canonical C-terminal PilZ domain. Dissection of the domains demonstrated that the separated N-terminal domain bound nucleic acids independently of c-di-GMP. The C-terminal domain, which includes the c-di-GMP binding motifs, did not bind nucleic acids under any tested conditions. Our data are supported by computational docking, which predicts that c-di-GMP binding at the C-terminal domain stabilizes the overall protein structure and facilitates PlzA-DNA interactions via residues in the N-terminal domain. Based on our data, we propose that levels of c-di-GMP during the various stages of the enzootic life cycle direct PlzA binding to regulatory targets.

The molecular determinants of classical pathway complement inhibition by OspEF-related proteins of Borrelia burgdorferi.

The complement system serves as the first line of defense against invading pathogens by promoting opsonophagocytosis and bacteriolysis. Antibody-dependent activation of complement occurs through the classical pathway and relies on the activity of initiating complement proteases of the C1 complex, C1r and C1s. The causative agent of Lyme disease, Borrelia burgdorferi, expresses two paralogous outer surface lipoproteins of the OspEF-related protein family, ElpB and ElpQ, that act as specific inhibitors of classical pathway activation. We have previously shown that ElpB and ElpQ bind directly to C1r and C1s with high affinity and specifically inhibit C2 and C4 cleavage by C1s. To further understand how these novel protease inhibitors function, we carried out a series of hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments using ElpQ and full-length activated C1s as a model of Elp-protease interaction. Comparison of HDX-MS profiles between unbound ElpQ and the ElpQ/C1s complex revealed a putative C1s-binding site on ElpQ. HDX-MS-guided, site-directed ElpQ mutants were generated and tested for direct binding to C1r and C1s using surface plasmon resonance. Several residues within the C-terminal region of ElpQ were identified as important for protease binding, including a single conserved tyrosine residue that was required for ElpQ- and ElpB-mediated complement inhibition. Collectively, our study identifies key molecular determinants for classical pathway protease recognition by Elp proteins. This investigation improves our understanding of the unique complement inhibitory mechanism employed by Elp proteins which serve as part of a sophisticated complement evasion system present in Lyme disease spirochetes.

A Fur family protein BosR is a novel RNA-binding protein that controls rpoS RNA stability in the Lyme disease pathogen.

The σ54-σS sigma factor cascade plays a central role in regulating differential gene expression during the enzootic cycle of Borreliella burgdorferi, the Lyme disease pathogen. In this pathway, the primary transcription of rpoS (which encodes σS) is under the control of σ54 which is activated by a bacterial enhancer-binding protein (EBP), Rrp2. The σ54-dependent activation in B. burgdorferi has long been thought to be unique, requiring an additional factor, BosR, a homologue of classical Fur/PerR repressor/activator. However, how BosR is involved in this σ54-dependent activation remains unclear and perplexing. In this study, we demonstrate that BosR does not function as a regulator for rpoS transcriptional activation. Instead, it functions as a novel RNA-binding protein that governs the turnover rate of rpoS mRNA. We further show that BosR directly binds to the 5' untranslated region (UTR) of rpoS mRNA, and the binding region overlaps with a region required for rpoS mRNA degradation. Mutations within this 5'UTR region result in BosR-independent RpoS production. Collectively, these results uncover a novel role of Fur/PerR family regulators as RNA-binding proteins and redefine the paradigm of the σ54-σS pathway in B. burgdorferi.

BadR directly represses the expression of the glycerol utilization operon in the Lyme disease pathogen.

Glycerol utilization as a carbohydrate source by Borreliella burgdorferi, the Lyme disease spirochete, is critical for its successful colonization and persistence in the tick vector. The expression of the glpFKD (glp) operon, which encodes proteins for glycerol uptake/utilization, must be tightly regulated during the enzootic cycle of B. burgdorferi. Previous studies have established that the second messenger cyclic di-GMP (c-di-GMP) is required for the activation of glp expression, while an alternative sigma factor RpoS acts as a negative regulator for glp expression. In the present study, we report identification of a cis element within the 5´ untranslated region of glp that exerts negative regulation of glp expression. Further genetic screen of known and predicted DNA-binding proteins encoded in the genome of B. burgdorferi uncovered that overexpressing Borrelia host adaptation regulator (BadR), a known global regulator, dramatically reduced glp expression. Similarly, the badR mutant significantly increased glp expression. Subsequent electrophoretic mobility shift assay analyses demonstrated that BadR directly binds to this cis element, thereby repressing glp independent of RpoS-mediated repression. The efficiency of BadR binding was further assessed in the presence of c-di-GMP and various carbohydrates. This finding highlights multi-layered positive and negative regulatory mechanisms employed by B. burgdorferi to synchronize glp expression throughout its enzootic cycle.IMPORTANCEBorreliella burgdorferi, the Lyme disease pathogen, must modulate its gene expression differentially to adapt successfully to its two disparate hosts. Previous studies have demonstrated that the glycerol uptake and utilization operon, glpFKD, plays a crucial role in spirochetal survival within ticks. However, the glpFKD expression must be repressed when B. burgdorferi transitions to the mammalian host. In this study, we identified a specific cis element responsible for the repression of glpFKD. We further pinpointed Borrelia host adaptation regulator as the direct binding protein to this cis element, thereby repressing glpFKD expression. This discovery paves the way for a deeper exploration of how zoonotic pathogens sense distinct hosts and switch their carbon source utilization during transmission.

Targeting Borrelia burgdorferi HtpG with a berserker molecule, a strategy for anti-microbial development.

Conventional antimicrobial discovery relies on targeting essential enzymes in pathogenic organisms, contributing to a paucity of new antibiotics to address resistant strains. Here, by targeting a non-essential enzyme, Borrelia burgdorferi HtpG, to deliver lethal payloads, we expand what can be considered druggable within any pathogen. We synthesized HS-291, an HtpG inhibitor tethered to the photoactive toxin verteporfin. Reactive oxygen species, generated by light, enables HS-291 to sterilize Borrelia cultures by causing oxidation of HtpG, and a discrete subset of proteins in proximity to the chaperone. This caused irreversible nucleoid collapse and membrane blebbing. Tethering verteporfin to the HtpG inhibitor was essential, since free verteporfin was not retained by Borrelia in contrast to HS-291. For this reason, we liken HS-291 to a berserker, wreaking havoc upon the pathogen's biology once selectively absorbed and activated. This strategy expands the druggable pathogenic genome and offsets antibiotic resistance by targeting non-essential proteins.

Structure of the Borrelia burgdorferi ATP-dependent metalloprotease FtsH in its functionally relevant hexameric form.

ATP-dependent proteases FtsH are conserved in bacteria, mitochondria, and chloroplasts, where they play an essential role in degradation of misfolded/unneeded membrane and cytosolic proteins. It has also been demonstrated that the FtsH homologous protein BB0789 is crucial for mouse and tick infectivity and in vitro growth of the Lyme disease-causing agent Borrelia burgdorferi. This is not surprising, considering B. burgdorferi complex life cycle, residing in both in mammals and ticks, which requires a wide range of membrane proteins and short-lived cytosolic regulatory proteins to invade and persist in the host organism. In the current study, we have solved the crystal structure of the cytosolic BB0789166-614, lacking both N-terminal transmembrane α-helices and the small periplasmic domain. The structure revealed the arrangement of the AAA+ ATPase and the zinc-dependent metalloprotease domains in a hexamer ring, which is essential for ATPase and proteolytic activity. The AAA+ domain was found in an ADP-bound state, while the protease domain showed coordination of a zinc ion by two histidine residues and one aspartic acid residue. The loop region that forms the central pore in the oligomer was poorly defined in the crystal structure and therefore predicted by AlphaFold to complement the missing structural details, providing a complete picture of the functionally relevant hexameric form of BB0789. We confirmed that BB0789 is functionally active, possessing both protease and ATPase activities, thus providing novel structural-functional insights into the protein, which is known to be absolutely necessary for B. burgdorferi to survive and cause Lyme disease.

The Rrp2-RpoN-RpoS pathway plays an important role in the blood-brain barrier transmigration of the Lyme disease pathogen.

Lyme disease, caused by Borrelia (or Borreliella) burgdorferi, is a complex multisystemic disorder that includes Lyme neuroborreliosis resulting from the invasion of both the central and peripheral nervous systems. However, factors that enable the pathogen to cross the blood-brain barrier (BBB) and invade the central nervous system (CNS) are still not well understood. The objective of this study was to identify the B. burgdorferi factors required for BBB transmigration. We utilized a transwell BBB model based on human brain-microvascular endothelial cells and focused on investigating the Rrp2-RpoN-RpoS pathway, a central regulatory pathway that is essential for mammalian infection by B. burgdorferi. Our results demonstrated that the Rrp2-RpoN-RpoS pathway is crucial for BBB transmigration. Furthermore, we identified OspC, a major surface lipoprotein controlled by the Rrp2-RpoN-RpoS pathway, as a significant contributor to BBB transmigration. Constitutive production of OspC in a mutant defective in the Rrp2-RpoN-RpoS pathway did not rescue the impairment in BBB transmigration, indicating that this pathway controls additional factors for this process. Two other major surface lipoproteins controlled by this pathway, DbpA/B and BBK32, appeared to be dispensable for BBB transmigration. In addition, both the surface lipoprotein OspA and the Rrp1 pathway, which are required B. burgdorferi colonization in the tick vector, were found not required for BBB transmigration. Collectively, our findings using in vitro transwell assays uncover another potential role of the Rrp2-RpoN-RpoS pathway in BBB transmigration of B. burgdorferi and invasion into the CNS.

A unique borrelial protein facilitates microbial immune evasion.

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.

The Lyme disease spirochete, Borrelia burgdorferi, as a model vector-borne pathogen: insights on regulation of gene and protein expression.

The Lyme disease spirochete persists in nature through cycles between ticks and vertebrates. Although the spirochete interacts with numerous, distinct tissues and environmental conditions during its infectious cycle, Borrelia burgdorferi appears to possess a limited ability to sense its external environment. This apparent paradox is being resolved through detailed investigations of the molecular mechanisms through which B. burgdorferi controls production of virulence-associated factors such as the Erp outer surface proteins. The results have led to development of a model for how B. burgdorferi controls expression of its diverse proteins, wherein physiological and metabolic states that are unique to specific points in the infectious cycle trigger changes in gene and protein expression levels.

A Borrelia burgdorferi LptD homolog is required for flipping of surface lipoproteins through the spirochetal outer membrane.

Borrelia spirochetes are unique among diderm bacteria in their lack of lipopolysaccharide (LPS) in the outer membrane (OM) and their abundance of surface-exposed lipoproteins with major roles in transmission, virulence, and pathogenesis. Despite their importance, little is known about how surface lipoproteins are translocated through the periplasm and the OM. Here, we characterized Borrelia burgdorferi BB0838, a distant homolog of the OM LPS assembly protein LptD. Using a CRISPR interference approach, we showed that BB0838 is required for cell growth and envelope stability. Upon BB0838 knockdown, surface lipoprotein OspA was retained in the inner leaflet of the OM, as determined by its inaccessibility to in situ proteolysis but its presence in OM vesicles. The topology of the OM porin/adhesin P66 remained unaffected. Quantitative mass spectrometry of the B. burgdorferi membrane-associated proteome confirmed the selective periplasmic retention of surface lipoproteins under BB0838 knockdown conditions. Additional analysis identified a single in situ protease-accessible BB0838 peptide that mapped to a predicted ß-barrel surface loop. Alphafold Multimer modeled a B. burgdorferi LptB2 FGCAD complex spanning the periplasm. Together, this suggests that BB0838/LptDBb facilitates the essential terminal step in spirochetal surface lipoprotein secretion, using an orthologous OM component of a pathway that secretes LPS in proteobacteria.

c-di-GMP regulates activity of the PlzA RNA chaperone from the Lyme disease spirochete.

PlzA is a c-di-GMP-binding protein crucial for adaptation of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi during its enzootic life cycle. Unliganded apo-PlzA is important for vertebrate infection, while liganded holo-PlzA is important for survival in the tick; however, the biological function of PlzA has remained enigmatic. Here, we report that PlzA has RNA chaperone activity that is inhibited by c-di-GMP binding. Holo- and apo-PlzA bind RNA and accelerate RNA annealing, while only apo-PlzA can strand displace and unwind double-stranded RNA. Guided by the crystal structure of PlzA, we identified several key aromatic amino acids protruding from the N- and C-terminal domains that are required for RNA-binding and unwinding activity. Our findings illuminate c-di-GMP as a switch controlling the RNA chaperone activity of PlzA, and we propose that complex RNA-mediated modulatory mechanisms allow PlzA to regulate gene expression during both the vector and host phases of the B. burgdorferi life cycle.

Borrelia burgdorferi Outer Surface Protein C Is Not the Sole Determinant of Dissemination in Mammals.

Lyme disease in the United States is most often caused by Borrelia burgdorferi sensu stricto. After a tick bite, the patient may develop erythema migrans at that site. If hematogenous dissemination occurs, the patient may then develop neurologic manifestations, carditis, or arthritis. Host-pathogen interactions include factors that contribute to hematogenous dissemination to other body sites. Outer surface protein C (OspC), a surface-exposed lipoprotein of B. burgdorferi, is essential during the early stages of mammalian infection. There is a high degree of genetic variation at the ospC locus, and certain ospC types are more frequently associated with hematogenous dissemination in patients, suggesting that OspC may be a major contributing factor to the clinical outcome of B. burgdorferi infection. In order to evaluate the role of OspC in B. burgdorferi dissemination, ospC was exchanged between B. burgdorferi isolates with different capacities to disseminate in laboratory mice, and these strains were then tested for their ability to disseminate in mice. The results indicated that the ability of B. burgdorferi to disseminate in mammalian hosts does not depend on OspC alone. The complete genome sequences of two closely related strains of B. burgdorferi with differing dissemination phenotypes were determined, but a specific genetic locus that could explain the differences in the phenotypes could not be definitively identified. The animal studies performed clearly demonstrated that OspC is not the sole determinant of dissemination. Future studies of the type described here with additional borrelial strains will hopefully clarify the genetic elements associated with hematogenous dissemination.

Quantitative analyses of interactions between SpoVG and RNA/DNA.

The Borrelia burgdorferi SpoVG protein has previously been found to be a DNA- and RNA-binding protein. To aid in the elucidation of ligand motifs, affinities for numerous RNAs, ssDNAs, and dsDNAs were measured and compared. The loci used in the study were spoVG, glpFKD, erpAB, bb0242, flaB, and ospAB, with particular focus on the untranslated 5' portion of the mRNAs. Performing binding and competition assays yielded that the 5' end of spoVG mRNA had the highest affinity while the lowest observed affinity was to the 5' end of flaB mRNA. Mutagenesis studies of spoVG RNA and ssDNA sequences suggested that the formation of SpoVG-nucleic acid complexes are not entirely dependent on either sequence or structure. Additionally, exchanging uracil for thymine in ssDNAs did not affect protein-nucleic acid complex formation.

Pleomorphic Variants of Borreliella (syn. Borrelia) burgdorferi Express Evolutionary Distinct Transcriptomes.

Borreliella (syn. Borrelia) burgdorferi is a spirochete bacterium that causes tick-borne Lyme disease. Along its lifecycle B. burgdorferi develops several pleomorphic forms with unclear biological and medical relevance. Surprisingly, these morphotypes have never been compared at the global transcriptome level. To fill this void, we grew B. burgdorferi spirochete, round body, bleb, and biofilm-dominated cultures and recovered their transcriptomes by RNAseq profiling. We found that round bodies share similar expression profiles with spirochetes, despite their morphological differences. This sharply contrasts to blebs and biofilms that showed unique transcriptomes, profoundly distinct from spirochetes and round bodies. To better characterize differentially expressed genes in non-spirochete morphotypes, we performed functional, positional, and evolutionary enrichment analyses. Our results suggest that spirochete to round body transition relies on the delicate regulation of a relatively small number of highly conserved genes, which are located on the main chromosome and involved in translation. In contrast, spirochete to bleb or biofilm transition includes substantial reshaping of transcription profiles towards plasmids-residing and evolutionary young genes, which originated in the ancestor of Borreliaceae. Despite their abundance the function of these Borreliaceae-specific genes is largely unknown. However, many known Lyme disease virulence genes implicated in immune evasion and tissue adhesion originated in this evolutionary period. Taken together, these regularities point to the possibility that bleb and biofilm morphotypes might be important in the dissemination and persistence of B. burgdorferi inside the mammalian host. On the other hand, they prioritize the large pool of unstudied Borreliaceae-specific genes for functional characterization because this subset likely contains undiscovered Lyme disease pathogenesis genes.

Structural Elucidation of a Protective B Cell Epitope on Outer Surface Protein C (OspC) of the Lyme Disease Spirochete, Borreliella burgdorferi.

Outer surface protein C (OspC) plays a pivotal role in mediating tick-to-host transmission and infectivity of the Lyme disease spirochete, Borreliella burgdorferi. OspC is a helical-rich homodimer that interacts with tick salivary proteins, as well as components of the mammalian immune system. Several decades ago, it was shown that the OspC-specific monoclonal antibody, B5, was able to passively protect mice from experimental tick-transmitted infection by B. burgdorferi strain B31. However, B5's epitope has never been elucidated, despite widespread interest in OspC as a possible Lyme disease vaccine antigen. Here, we report the crystal structure of B5 antigen-binding fragments (Fabs) in complex with recombinant OspC type A (OspCA). Each OspC monomer within the homodimer was bound by a single B5 Fab in a side-on orientation, with contact points along OspC's α-helix 1 and α-helix 6, as well as interactions with the loop between α-helices 5 and 6. In addition, B5's complementarity-determining region (CDR) H3 bridged the OspC-OspC' homodimer interface, revealing the quaternary nature of the protective epitope. To provide insight into the molecular basis of B5 serotype specificity, we solved the crystal structures of recombinant OspC types B and K and compared them to OspCA. This study represents the first structure of a protective B cell epitope on OspC and will aid in the rational design of OspC-based vaccines and therapeutics for Lyme disease. IMPORTANCE The spirochete Borreliella burgdorferi is a causative agent of Lyme disease, the most common tickborne disease in the United States. The spirochete is transmitted to humans during the course of a tick taking a bloodmeal. After B. burgdorferi is deposited into the skin of a human host, it replicates locally and spreads systemically, often resulting in clinical manifestations involving the central nervous system, joints, and/or heart. Antibodies directed against B. burgdorferi's outer surface protein C (OspC) are known to block tick-to-host transmission, as well as dissemination of the spirochete within a mammalian host. In this report, we reveal the first atomic structure of one such antibody in complex with OspC. Our results have implications for the design of a Lyme disease vaccine capable of interfering with multiple stages in B. burgdorferi infection.

Erp and Rev Adhesins of the Lyme Disease Spirochete's Ubiquitous cp32 Prophages Assist the Bacterium during Vertebrate Infection.

Almost all spirochetes in the genus Borrelia (sensu lato) naturally contain multiple variants of closely related prophages. In the Lyme disease borreliae, these prophages are maintained as circular episomes that are called circular plasmid 32 kb (cp32s). The cp32s of Lyme agents are particularly unique in that they encode two distinct families of lipoproteins, namely, Erp and Rev, that are expressed on the bacterial outer surface during infection of vertebrate hosts. All identified functions of those outer surface proteins involve interactions between the spirochetes and host molecules, as follows: Erp proteins bind plasmin(ogen), laminin, glycosaminoglycans, and/or components of complement and Rev proteins bind fibronectin. Thus, cp32 prophages provide their bacterial hosts with surface proteins that can enhance infection processes, thereby facilitating their own survival. Horizontal transfer via bacteriophage particles increases the spread of beneficial alleles and creates diversity among Erp and Rev proteins.

Borrelia burgdorferi DnaA and the Nucleoid-Associated Protein EbfC Coordinate Expression of the dnaX-ebfC Operon.

Borrelia burgdorferi, the spirochete agent of Lyme disease, has evolved within a consistent infectious cycle between tick and vertebrate hosts. The transmission of the pathogen from tick to vertebrate is characterized by rapid replication and a change in the outer surface protein profile. EbfC, a highly conserved nucleoid-associated protein, binds throughout the borrelial genome, affecting expression of many genes, including the Erp outer surface proteins. In B. burgdorferi, like many other bacterial species, ebfC is cotranscribed with dnaX, an essential component of the DNA polymerase III holoenzyme, which facilitates chromosomal replication. The expression of the dnaX-ebfC operon is tied to the spirochete's replication rate, but the underlying mechanism for this connection was unknown. In this work, we provide evidence that the expression of dnaX-ebfC is controlled by direct interactions of DnaA, the chromosomal replication initiator, and EbfC at the unusually long dnaX-ebfC 5' untranslated region (UTR). Both proteins bind to the 5' UTR DNA, with EbfC also binding to the RNA. The DNA binding of DnaA to this region was similarly impacted by ATP and ADP. In vitro studies characterized DnaA as an activator of dnaX-ebfC and EbfC as an antiactivator. We further found evidence that DnaA may regulate other genes essential for replication. IMPORTANCE The dual life cycle of Borrelia burgdorferi, the causative agent of Lyme disease, is characterized by periods of rapid and slowed replication. The expression patterns of many of the spirochete's virulence factors are impacted by these changes in replication rates. The connection between replication and virulence can be understood at the dnaX-ebfC operon. DnaX is an essential component of the DNA polymerase III holoenzyme, which replicates the chromosome. EbfC is a nucleoid-associated protein that regulates the infection-associated outer surface Erp proteins, as well as other transcripts. The expression of dnaX-ebfC is tied to replication rate, which we demonstrate is mediated by DnaA, the master chromosomal initiator protein and transcription factor, and EbfC.

Increased macrophage phagocytic activity with TLR9 agonist conjugation of an anti- Borrelia burgdorferi monoclonal antibody.

Borrelia burgdorferi (Bb) infection causes Lyme disease, for which there is need for more effective therapies. Here, we sequenced the antibody repertoire of plasmablasts in Bb-infected humans. We expressed recombinant monoclonal antibodies (mAbs) representing the identified plasmablast clonal families, and identified their binding specificities. Our recombinant anti-Bb mAbs exhibit a range of activity in mediating macrophage phagocytosis of Bb. To determine if we could increase the macrophage phagocytosis-promoting activity of our anti-Bb mAbs, we generated a TLR9-agonist CpG-oligo-conjugated anti-BmpA mAb. We demonstrated that our CpG-conjugated anti-BmpA mAb exhibited increased peak Bb phagocytosis at 12-24 h, and sustained macrophage phagocytosis over 60+ hrs. Further, our CpG-conjugated anti-BmpA mAb induced macrophages to exhibit a sustained activation morphology. Our findings demonstrate the potential for TLR9-agonist CpG-oligo conjugates to enhance mAb-mediated clearance of Bb, and this approach might also enhance the activity of other anti-microbial mAbs.