Structure of a human monoclonal antibody in complex with Outer surface protein C (OspC) of the Lyme disease spirochete, Borreliella burgdorferi.

Lyme disease is a tick-borne, multisystem infection caused by the spirochete, Borreliella burgdorferi . Although antibodies have been implicated in the resolution of Lyme disease, the specific B cell epitopes targeted during human infections remain largely unknown. In this study, we characterized and defined the structural epitope of a patient-derived bactericidal monoclonal IgG ("B11") against Outer surface protein C (OspC), a homodimeric lipoprotein necessary for B. burgdorferi tick-mediated transmission and early-stage colonization of vertebrate hosts. High-resolution epitope mapping was accomplished through hydrogen deuterium exchange-mass spectrometry (HDX-MS) and X-ray crystallography. Structural analysis of B11 Fab-OspC A complexes revealed the B11 Fabs associated in a 1:1 stoichiometry with the lateral faces of OspC A homodimers such that the antibodies are essentially positioned perpendicular to the spirochete's outer surface. B11's primary contacts reside within the membrane proximal regions of α-helices 1 and 6 and adjacent loops 5 and 6 in one OspC A monomer. In addition, B11 spans the OspC A dimer interface, engaging opposing α-helix 1', α-helix 2', and loop 2-3' in the second OspC A monomer. The B11-OspC A structure is reminiscent of the recently solved mouse transmission blocking monoclonal IgG B5 in complex with OspC A , indicating a mode of engagement with OspC that is conserved across species. In conclusion, we provide the first detailed insight into the interaction between a functional human antibody and an immunodominant Lyme disease antigen long considered an important vaccine target.

Single-domain antibodies reveal unique borrelicidal epitopes on the Lyme disease vaccine antigen, outer surface protein A (OspA).

Camelid-derived, single-domain antibodies (VHHs) have proven to be extremely powerful tools in defining the antigenic landscape of immunologically heterogeneous surface proteins. In this report, we generated a phage-displayed VHH library directed against the candidate Lyme disease vaccine antigen, outer surface protein A (OspA). Two alpacas were immunized with recombinant OspA serotype 1 from Borrelia burgdorferi sensu stricto strain B31, in combination with the canine vaccine RECOMBITEK Lyme containing lipidated OspA. The phage library was subjected to two rounds of affinity enrichment ("panning") against recombinant OspA, yielding 21 unique VHHs within two epitope bins, as determined through competition enzyme linked immunosorbent assays (ELISAs) with a panel of OspA-specific human monoclonal antibodies. Epitope refinement was conducted by hydrogen exchange-mass spectrometry. Six of the monovalent VHHs were expressed as human IgG1-Fc fusion proteins and shown to have functional properties associated with protective human monoclonal antibodies, including B. burgdorferi agglutination, outer membrane damage, and complement-dependent borreliacidal activity. The VHHs displayed unique reactivity profiles with the seven OspA serotypes associated with B. burgdorferi genospecies in the United States and Europe consistent with there being unique epitopes across OspA serotypes that should be considered when designing and evaluating multivalent Lyme disease vaccines.

Structure of a transmission blocking antibody in complex with Outer surface protein A from the Lyme disease spirochete, Borreliella burgdorferi.

319-44 is a human monoclonal antibody capable of passively protecting mice against tick-mediated infection with Borreliella burgdorferi, the bacterial genospecies responsible for Lyme disease in North America. In vitro, 319-44 has complement-dependent borreliacidal activity and spirochete agglutinating properties. Here, we report the 2.2 Å-resolution crystal structure of 319-44 Fab fragments in complex with Outer surface protein A (OspA), the ~30 kDa lipoprotein that was the basis of the first-generation Lyme disease vaccine approved in the United States. The 319-44 epitope is focused on OspA ß-strands 19, 20, and 21, and the loops between ß-strands 16-17, 18-19, and 20-21. Contact with loop 20-21 explains competition with LA-2, the murine monoclonal antibody used to estimate serum borreliacidal activities in the first-generation Lyme disease vaccine clinical trials. A high-resolution B-cell epitope map of OspA will accelerate structure-based design of second generation OspA-based vaccines.

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.

Methylation of rRNA as a host defense against rampant group II intron retrotransposition.

BACKGROUND: Group II introns are mobile retroelements, capable of invading new sites in DNA. They are self-splicing ribozymes that complex with an intron-encoded protein to form a ribonucleoprotein that targets DNA after splicing. These molecules can invade DNA site-specifically, through a process known as retrohoming, or can invade ectopic sites through retrotransposition. Retrotransposition, in particular, can be strongly influenced by both environmental and cellular factors. RESULTS: To investigate host factors that influence retrotransposition, we performed random insertional mutagenesis using the ISS1 transposon to generate a library of over 1000 mutants in Lactococcus lactis, the native host of the Ll.LtrB group II intron. By screening this library, we identified 92 mutants with increased retrotransposition frequencies (RTP-ups). We found that mutations in amino acid transport and metabolism tended to have increased retrotransposition frequencies. We further explored a subset of these RTP-up mutants, the most striking of which is a mutant in the ribosomal RNA methyltransferase rlmH, which exhibited a reproducible 20-fold increase in retrotransposition frequency. In vitro and in vivo experiments revealed that ribosomes in the rlmH mutant were defective in the m3Ψ modification and exhibited reduced binding to the intron RNA. CONCLUSIONS: Taken together, our results reinforce the importance of the native host organism in regulating group II intron retrotransposition. In particular, the evidence from the rlmH mutant suggests a role for ribosome modification in limiting rampant retrotransposition.

Group II intron as cold sensor for self-preservation and bacterial conjugation.

Group II introns are self-splicing ribozymes and mobile genetic elements. Splicing is required for both expression of the interrupted host gene and intron retromobility. For the pRS01 plasmid-encoded Lactococcus lactis group II intron, Ll.LtrB, splicing enables expression of the intron's host relaxase protein. Relaxase, in turn, initiates horizontal transfer of the conjugative pRS01 plasmid and stimulates retrotransposition of the intron. Little is known about how splicing of bacterial group II introns is influenced by environmental conditions. Here, we show that low temperatures can inhibit Ll.LtrB intron splicing. Whereas autocatalysis is abolished in the cold, splicing is partially restored by the intron-encoded protein (IEP). Structure profiling reveals cold-induced disruptions of key tertiary interactions, suggesting that a kinetic trap prevents the intron RNA from assuming its native state. Interestingly, while reduced levels of transcription and splicing lead to a paucity of excised intron in the cold, levels of relaxase mRNA are maintained, partially due to diminished intron-mediated mRNA targeting, allowing intron spread by conjugal transfer. Taken together, this study demonstrates not only the intrinsic cold sensitivity of group II intron splicing and the role of the IEP for cold-stress adaptation, but also maintenance of horizontal plasmid and intron transfer under cold-shock.

Group II intron inhibits conjugative relaxase expression in bacteria by mRNA targeting.

Group II introns are mobile ribozymes that are rare in bacterial genomes, often cohabiting with various mobile elements, and seldom interrupting housekeeping genes. What accounts for this distribution has not been well understood. Here, we demonstrate that Ll.LtrB, the group II intron residing in a relaxase gene on a conjugative plasmid from Lactococcus lactis, inhibits its host gene expression and restrains the naturally cohabiting mobile element from conjugative horizontal transfer. We show that reduction in gene expression is mainly at the mRNA level, and results from the interaction between exon-binding sequences (EBSs) in the intron and intron-binding sequences (IBSs) in the mRNA. The spliced intron targets the relaxase mRNA and reopens ligated exons, causing major mRNA loss. Taken together, this study provides an explanation for the distribution and paucity of group II introns in bacteria, and suggests a potential force for those introns to evolve into spliceosomal introns.

Structural accommodations accompanying splicing of a group II intron RNP.

Group II introns, the putative progenitors of spliceosomal introns and retrotransposons, are ribozymes that are capable of self-splicing and DNA invasion. In the cell, group II introns form ribonucleoprotein (RNP) complexes with an intron-encoded protein, which is essential to folding, splicing and retromobility of the intron. To understand the structural accommodations underlying splicing, in preparation for retromobility, we probed the endogenously expressed Lactococcus lactis Ll.LtrB group II intron RNP using SHAPE. The results, which are consistent in vivo and in vitro, provide insights into the dynamics of the intron RNP as well as RNA-RNA and RNA-protein interactions. By comparing the excised intron RNP with mutant RNPs in the precursor state, confined SHAPE profile differences were observed, indicative of rearrangements at the active site as well as disengagement at the functional RNA-protein interface in transition between the two states. The exon-binding sequences in the intron RNA, which interact with the 5' exon and the target DNA, show increased flexibility after splicing. In contrast, stability of major tertiary and protein interactions maintains the scaffold of the RNA through the splicing transition, while the active site is realigned in preparation for retromobility.

Structure of a group II intron in complex with its reverse transcriptase.

Bacterial group II introns are large catalytic RNAs related to nuclear spliceosomal introns and eukaryotic retrotransposons. They self-splice, yielding mature RNA, and integrate into DNA as retroelements. A fully active group II intron forms a ribonucleoprotein complex comprising the intron ribozyme and an intron-encoded protein that performs multiple activities including reverse transcription, in which intron RNA is copied into the DNA target. Here we report cryo-EM structures of an endogenously spliced Lactococcus lactis group IIA intron in its ribonucleoprotein complex form at 3.8-Å resolution and in its protein-depleted form at 4.5-Å resolution, revealing functional coordination of the intron RNA with the protein. Remarkably, the protein structure reveals a close relationship between the reverse transcriptase catalytic domain and telomerase, whereas the active splicing center resembles the spliceosomal Prp8 protein. These extraordinary similarities hint at intricate ancestral relationships and provide new insights into splicing and retromobility.

SufB intein of Mycobacterium tuberculosis as a sensor for oxidative and nitrosative stresses.

Inteins are mobile genetic elements that self-splice at the protein level. Mycobacteria have inteins inserted into several important genes, including those corresponding to the iron-sulfur cluster assembly protein SufB. Curiously, the SufB inteins are found primarily in mycobacterial species that are potential human pathogens. Here we discovered an exceptional sensitivity of Mycobacterium tuberculosis SufB intein splicing to oxidative and nitrosative stresses when expressed in Escherichia coli. This effect results from predisposition of the intein's catalytic cysteine residues to oxidative and nitrosative modifications. Experiments with a fluorescent reporter system revealed that reactive oxygen species and reactive nitrogen species inhibit SufB extein ligation by forcing either precursor accumulation or N-terminal cleavage. We propose that splicing inhibition is an immediate, posttranslational regulatory response that can be either reversible, by inducing precursor accumulation, or irreversible, by inducing N-terminal cleavage, which may potentially channel mycobacteria into dormancy under extreme oxidative and nitrosative stresses.

RNA-RNA interactions and pre-mRNA mislocalization as drivers of group II intron loss from nuclear genomes.

Group II introns are commonly believed to be the progenitors of spliceosomal introns, but they are notably absent from nuclear genomes. Barriers to group II intron function in nuclear genomes therefore beg examination. A previous study showed that nuclear expression of a group II intron in yeast results in nonsense-mediated decay and translational repression of mRNA, and that these roadblocks to expression are group II intron-specific. To determine the molecular basis for repression of gene expression, we investigated cellular dynamics of processed group II intron RNAs, from transcription to cellular localization. Our data show pre-mRNA mislocalization to the cytoplasm, where the RNAs are targeted to foci. Furthermore, tenacious mRNA-pre-mRNA interactions, based on intron-exon binding sequences, result in reduced abundance of spliced mRNAs. Nuclear retention of pre-mRNA prevents this interaction and relieves these expression blocks. In addition to providing a mechanistic rationale for group II intron-specific repression, our data support the hypothesis that RNA silencing of the host gene contributed to expulsion of group II introns from nuclear genomes and drove the evolution of spliceosomal introns.

Group II intron-ribosome association protects intron RNA from degradation.

The influence of the cellular environment on the structures and properties of catalytic RNAs is not well understood, despite great interest in ribozyme function. Here we report on ribosome association of group II introns, which are ribozymes that are important because of their putative ancestry to spliceosomal introns and retrotransposons, their retromobility via an RNA intermediate, and their application as gene delivery agents. We show that group II intron RNA, in complex with the intron-encoded protein from the native Lactoccocus lactis host, associates strongly with ribosomes in vivo. Ribosomes have little effect on intron ribozyme activities; rather, the association with host ribosomes protects the intron RNA against degradation by RNase E, an enzyme previously shown to be a silencer of retromobility in Escherichia coli. The ribosome interacts strongly with the intron, exerting protective effects in vivo and in vitro, as demonstrated by genetic and biochemical experiments. These results are consistent with the ribosome influencing the integrity of catalytic RNAs in bacteria in the face of degradative nucleases that regulate intron mobility.

Global regulators orchestrate group II intron retromobility.

Group II introns are hypothesized to share common ancestry with both nuclear spliceosomal introns and retrotransposons, which collectively occupy the majority of genome space in higher eukaryotes. These phylogenetically diverse introns are mobile retroelements that move through an RNA intermediate. Disruption of Escherichia coli genes encoding enzymes that catalyze synthesis of global regulators cAMP and ppGpp inhibits group II intron retromobility. These small molecules program genetic transitions between nutrient excess and starvation. Accordingly, we demonstrated that glucose depletion of wild-type cells and cAMP supplementation of mutants stimulated retromobility. Likewise, amino acid starvation, which induces the alarmone ppGpp, activated retromobility. In both cases, retrotransposition to ectopic sites was favored over retrohoming. Interestingly, these stimulatory effects are mediated at the level of the DNA target, rather than of expression of the retroelement. Thereby, during metabolic stress, cAMP and ppGpp control group II intron movement in concert with the cell's global genetic circuitry, stimulating genetic diversity.

A mutant screen reveals RNase E as a silencer of group II intron retromobility in Escherichia coli.

Group II introns are mobile retroelements that invade their hosts. The Lactococcus lactis group II intron recruits cellular polymerases, nucleases, and DNA ligase to complete the retromobility process in Escherichia coli. Here we describe a genetic screen with a Tn5 transposon library to identify other E. coli functions involved in retromobility of the L. lactis LtrB intron. Thirteen disruptions that reproducibly resulted in increased or decreased retrohoming levels into the E. coli chromosome were isolated. These functions were classified as factors involved in RNA processing, DNA replication, energy metabolism, and global regulation. Here we characterize a novel mutant in the rne promoter region, which regulates RNase E expression. Retrohoming and retrotransposition levels are elevated in the rneTn5 mutant. The stimulatory effect of the mutation on retromobility results from intron RNA accumulation in the RNase E mutant. These results suggest that RNase E, which is the central component of the RNA degradosome, could regulate retrohoming levels in response to cellular physiology.

Bipolar localization of the group II intron Ll.LtrB is maintained in Escherichia coli deficient in nucleoid condensation, chromosome partitioning and DNA replication.

Group II introns are mobile genetic elements that invade their cognate intron-minus alleles via an RNA intermediate, in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. In Escherichia coli, retrotransposition of the lactococcal group II intron, Ll.LtrB, occurs preferentially within the Ori and Ter macrodomains of the E. coli chromosome. These macrodomains migrate towards the poles of the cell, where the intron-encoded protein, LtrA, localizes. Here we investigate whether alteration of nucleoid condensation, chromosome partitioning and replication affect retrotransposition frequencies, as well as bipolar localization of the Ll.LtrB intron integration and LtrA distribution in E. coli. We thus examined these properties in the absence of the nucleoid-associated proteins H-NS, StpA and MukB, in variants of partitioning functions including the centromere-like sequence migS and the actin homologue MreB, as well as in the replication mutants DeltaoriC, seqA, tus and topoIV (ts). Although there were some dramatic fluctuations in retrotransposition levels in these hosts, bipolar localization of integration events was maintained. LtrA was consistently found in nucleoid-free regions, with its localization to the cellular poles being largely preserved in these hosts. Together, these results suggest that bipolar localization of group II intron retrotransposition results from the residence of the intron-encoded protein at the poles of the cell.

Retrotransposition strategies of the Lactococcus lactis Ll.LtrB group II intron are dictated by host identity and cellular environment.

Group II introns are mobile retroelements that invade their cognate intron-minus gene in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. Previous studies of the Lactococcus lactis intron Ll.LtrB indicated that in its native host, as in Escherichia coli, retrohoming occurs by the intron RNA reverse splicing into double-stranded DNA (dsDNA) through an endonuclease-dependent pathway. However, in retrotransposition in L. lactis, the intron inserts predominantly into single-stranded DNA (ssDNA), in an endonuclease-independent manner. This work describes the retrotransposition of the Ll.LtrB intron in E. coli, using a retrotransposition indicator gene previously employed in our L. lactis studies. Unlike in L. lactis, in E. coli, Ll.LtrB retrotransposed frequently into dsDNA, and the process was dependent on the endonuclease activity of the intron-encoded protein. Further, the endonuclease-dependent insertions preferentially occurred around the origin and terminus of chromosomal DNA replication. Insertions in E. coli can also occur through an endonuclease-independent pathway, and, as in L. lactis, such events have a more random integration pattern. Together these findings show that Ll.LtrB can retrotranspose through at least two distinct mechanisms and that the host environment influences the choice of integration pathway. Additionally, growth conditions affect the insertion pattern. We propose a model in which DNA replication, compactness of the nucleoid and chromosomal localization influence target site preference.