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.

Lipoproteome screening of the Lyme disease agent identifies inhibitors of antibody-mediated complement killing.

Spirochetal pathogens, such as the causative agent of Lyme disease, Borrelia burgdorferi sensu lato, encode an abundance of lipoproteins; however, due in part to their evolutionary distance from more well-studied bacteria, such as Proteobacteria and Firmicutes, few spirochetal lipoproteins have assigned functions. Indeed, B. burgdorferi devotes almost 8% of its genome to lipoprotein genes and interacts with its environment primarily through the production of at least 80 surface-exposed lipoproteins throughout its tick vector­vertebrate host lifecycle. Several B. burgdorferi lipoproteins have been shown to serve roles in cellular adherence or immune evasion, but the functions for most B. burgdorferi surface lipoproteins remain unknown. In this study, we developed a B. burgdorferi lipoproteome screening platform utilizing intact spirochetes that enables the identification of previously unrecognized host interactions. As spirochetal survival in the bloodstream is essential for dissemination, we targeted our screen to C1, the first component of the classical (antibody-initiated) complement pathway. We identified two high-affinity C1 interactions by the paralogous lipoproteins, ElpB and ElpQ (also termed ErpB and ErpQ, respectively). Using biochemical, microbiological, and biophysical approaches, we demonstrate that ElpB and ElpQ bind the activated forms of the C1 proteases, C1r and C1s, and represent a distinct mechanistic class of C1 inhibitors that protect the spirochete from antibody-mediated complement killing. In addition to identifying a mode of complement inhibition, our study establishes a lipoproteome screening methodology as a discovery platform for identifying direct host­pathogen interactions that are central to the pathogenesis of spirochetes, such as the Lyme disease agent.

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.

Inducible CRISPRi-Based Operon Silencing and Selective in Trans Gene Complementation in Borrelia burgdorferi.

To accelerate genetic studies on the Lyme disease pathogen Borrelia burgdorferi, we developed an enhanced CRISPR interference (CRISPRi) approach for isopropyl-ß-d-thiogalactopyranoside (IPTG)-inducible repression of specific B. burgdorferi genes. The entire system is encoded on a compact 11-kb shuttle vector plasmid that allows for inducible expression of both the sgRNA module and a nontoxic codon-optimized dCas9 protein. We validated this CRISPRi system by targeting the genes encoding OspA and OspB, abundant surface lipoproteins coexpressed by a single operon, and FlaB, the major subunit forming the periplasmic flagella. As in other systems, single guide RNAs (sgRNAs) complementary to the nontemplate strand were consistently effective in gene repression, with 4- to 994-fold reductions in targeted transcript levels and concomitant reductions of protein levels. Furthermore, we showed that ospAB knockdowns could be selectively complemented in trans for OspA expression via the insertion of CRISPRi-resistant, synonymously or nonsynonymously mutated protospacer adjacent motif (PAM*) ospA alleles into a unique site within the CRISPRi plasmid. Together, this establishes CRISPRi PAM* as a robust new genetic tool to simplify the study of B. burgdorferi genes, bypassing the need for gene disruptions by allelic exchange and avoiding rare codon toxicity from the heterologous expression of dCas9. IMPORTANCE Borrelia burgdorferi, the spirochetal bacterium causing Lyme disease, is a tick-borne pathogen of global importance. Here, we expand the genetic toolbox for studying B. burgdorferi physiology and pathogenesis by establishing a single plasmid-based, fully inducible, and nontoxic CRISPR interference (CRISPRi) system for transcriptional silencing of B. burgdorferi genes and operons. We also show that alleles of CRISPRi-targeted genes with mutated protospacer-adjacent motif (PAM*) sites are CRISPRi resistant and can be used for simultaneous in trans gene complementation. The CRISPRi PAM* system will streamline the study of essential Borrelia proteins and accelerate investigations into their structure-function relationships.

Endogenous Linear Plasmids lp28-4 and lp25 Are Required for Infectivity and Restriction Protection in the Lyme Disease Spirochete Borrelia mayonii.

Borrelia mayonii is a newly recognized causative agent of Lyme disease in the Upper Midwestern United States, with distinct clinical presentations compared to classical Lyme disease caused by other Lyme Borrelia species. However, little is known about the B. mayonii genetic determinants required for establishing infection or perpetuating disease in mammals. Extrachromosomal plasmids in Borrelia species often encode proteins necessary for infection and pathogenesis, and spontaneous loss of these plasmids can lead to the identification of virulence determinant genes. Here, we describe infection of Lyme disease-susceptible C3H mice with B. mayonii, and show bacterial dissemination and persistence in peripheral tissues. Loss of endogenous plasmids, including lp28-4, lp25, and lp36 correlated with reduced infectivity in mice. The apparent requirement for lp28-4 during murine infection suggests the presence of a novel virulence determinant, as this plasmid does not encode homologs of any known virulence determinant. We also describe transformation and stable maintenance of a self-replicating shuttle vector in B. mayonii, and show that loss of either lp25 or lp28-4 correlated with increased transformation competency. Finally, we demonstrate that linear plasmids lp25 and lp28-4 each encode functional restriction modification systems with distinct but partially overlapping target modification sequences, which likely accounts for the observed decrease in transformation efficiency when those plasmids are present. Taken together, this study describes a role for endogenous plasmids in mammalian infection and restriction protection in the Lyme disease spirochete Borrelia mayonii.

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.

The Lyme disease spirochete's BpuR DNA/RNA-binding protein is differentially expressed during the mammal-tick infectious cycle, which affects translation of the SodA superoxide dismutase.

When the Lyme disease spirochete, Borrelia burgdorferi, transfers from a feeding tick into a human or other vertebrate host, the bacterium produces vertebrate-specific proteins and represses factors needed for arthropod colonization. Previous studies determined that the B. burgdorferi BpuR protein binds to its own mRNA and autoregulates its translation, and also serves as co-repressor of erp transcription. Here, we demonstrate that B. burgdorferi controls transcription of bpuR, expressing high levels of bpuR during tick colonization but significantly less during mammalian infection. The master regulator of chromosomal replication, DnaA, was found to bind specifically to a DNA sequence that overlaps the bpuR promoter. Cultured B. burgdorferi that were genetically manipulated to produce elevated levels of BpuR exhibited altered levels of several proteins, although BpuR did not impact mRNA levels. Among these was the SodA superoxide dismutase, which is essential for mammalian infection. BpuR bound to sodA mRNA in live B. burgdorferi, and a specific BpuR-binding site was mapped 5' of the sodA open reading frame. Recognition of posttranscriptional regulation of protein levels by BpuR adds another layer to our understanding of the B. burgdorferi regulome, and provides further evidence that bacterial protein levels do not always correlate directly with mRNA levels.

Spirochetal Lipoproteins in Pathogenesis and Immunity.

Lipoproteins are lipid-modified proteins that dominate the spirochetal proteome. While found in all bacteria, spirochetal lipoproteins have unique features and play critical roles in spirochete biology. For this reason, considerable effort has been devoted to determining how the lipoproteome is generated. Essential features of the structural elements of lipoproteins are now understood with greater clarity, enabling greater confidence in identification of lipoproteins from genomic sequences. The journey from the ribosome to the outer membrane, and in some cases, to the cellular surface has been defined, including secretion, lipidation, sorting, and export across the outer membrane. Given their abundance and importance, it is not surprising that spirochetes have developed a number of strategies for regulating the spatiotemporal expression of lipoproteins. In some cases, lipoprotein expression is tied to various environmental cues, while in other cases, it is linked to growth rate. This regulation enables spirochetes to express certain lipoproteins at high levels in one phase of the spirochete lifecycle, while dramatically downregulating the same lipoproteins in other phases. The mammalian host has developed specialized mechanisms for recognizing lipoproteins and triggering an immune response. Evasion of that immune response is essential for spirochete persistence. For this reason, spirochetes have developed mechanisms for altering lipoproteins. Lipoproteins recognized by antibodies formed during infection are key serodiagnostic antigens. In addition, lipoprotein vaccines have been developed for generating an immune response to control or prevent a spirochete infection. This chapter summarizes our current understanding of lipoproteins in interactions of spirochetes with their hosts.

Comprehensive Spatial Analysis of the Borrelia burgdorferi Lipoproteome Reveals a Compartmentalization Bias toward the Bacterial Surface.

The Lyme disease spirochete Borrelia burgdorferi is unique among bacteria in its large number of lipoproteins that are encoded by a small, exceptionally fragmented, and predominantly linear genome. Peripherally anchored in either the inner or outer membrane and facing either the periplasm or the external environment, these lipoproteins assume varied roles. A prominent subset of lipoproteins functioning as the apparent linchpins of the enzootic tick-vertebrate infection cycle have been explored as vaccine targets. Yet, most of the B. burgdorferi lipoproteome has remained uncharacterized. Here, we comprehensively and conclusively localize the B. burgdorferi lipoproteome by applying established protein localization assays to a newly generated epitope-tagged lipoprotein expression library and by validating the obtained individual protein localization results using a sensitive global mass spectrometry approach. The derived consensus localization data indicate that 86 of the 125 analyzed lipoproteins encoded by B. burgdorferi are secreted to the bacterial surface. Thirty-one of the remaining 39 periplasmic lipoproteins are retained in the inner membrane, with only 8 lipoproteins being anchored in the periplasmic leaflet of the outer membrane. The localization of 10 lipoproteins was further defined or revised, and 52 surface and 23 periplasmic lipoproteins were newly localized. Cross-referencing prior studies revealed that the borrelial surface lipoproteome contributing to the host-pathogen interface is encoded predominantly by plasmids. Conversely, periplasmic lipoproteins are encoded mainly by chromosomal loci. These studies close a gap in our understanding of the functional lipoproteome of an important human pathogen and set the stage for more in-depth studies of thus-far-neglected spirochetal lipoproteins.IMPORTANCE The small and exceptionally fragmented genome of the Lyme disease spirochete Borrelia burgdorferi encodes over 120 lipoproteins. Studies in the field have predominantly focused on a relatively small number of surface lipoproteins that play important roles in the transmission and pathogenesis of this global human pathogen. Yet, a comprehensive spatial assessment of the entire borrelial lipoproteome has been missing. The current study newly identifies 52 surface and 23 periplasmic lipoproteins. Overall, two-thirds of the B. burgdorferi lipoproteins localize to the surface, while outer membrane lipoproteins facing the periplasm are rare. This analysis underscores the dominant contribution of lipoproteins to the spirochete's rather complex and adaptable host-pathogen interface, and it encourages further functional exploration of its lipoproteome.

Hybrid polarizing solids for pure hyperpolarized liquids through dissolution dynamic nuclear polarization.

Hyperpolarization of substrates for magnetic resonance spectroscopy (MRS) and imaging (MRI) by dissolution dynamic nuclear polarization (D-DNP) usually involves saturating the ESR transitions of polarizing agents (PAs; e.g., persistent radicals embedded in frozen glassy matrices). This approach has shown enormous potential to achieve greatly enhanced nuclear spin polarization, but the presence of PAs and/or glassing agents in the sample after dissolution can raise concerns for in vivo MRI applications, such as perturbing molecular interactions, and may induce the erosion of hyperpolarization in spectroscopy and MRI. We show that D-DNP can be performed efficiently with hybrid polarizing solids (HYPSOs) with 2,2,6,6-tetramethyl-piperidine-1-oxyl radicals incorporated in a mesostructured silica material and homogeneously distributed along its pore channels. The powder is wetted with a solution containing molecules of interest (for example, metabolites for MRS or MRI) to fill the pore channels (incipient wetness impregnation), and DNP is performed at low temperatures in a very efficient manner. This approach allows high polarization without the need for glass-forming agents and is applicable to a broad range of substrates, including peptides and metabolites. During dissolution, HYPSO is physically retained by simple filtration in the cryostat of the DNP polarizer, and a pure hyperpolarized solution is collected within a few seconds. The resulting solution contains the pure substrate, is free from any paramagnetic or other pollutants, and is ready for in vivo infusion.

The leptospiral outer membrane.

The outer membrane (OM) is the front line of leptospiral interactions with their environment and the mammalian host. Unlike most invasive spirochetes, pathogenic leptospires must be able to survive in both free-living and host-adapted states. As organisms move from one set of environmental conditions to another, the OM must cope with a series of conflicting challenges. For example, the OM must be porous enough to allow nutrient uptake, yet robust enough to defend the cell against noxious substances. In the host, the OM presents a surface decorated with adhesins and receptors for attaching to, and acquiring, desirable host molecules such as the complement regulator, Factor H.Factor H. On the other hand, the OM must enable leptospires to evade detection by the host's immune system on their way from sites of invasion through the bloodstream to the protected niche of the proximal tubule. The picture that is emerging of the leptospiral OM is that, while it shares many of the characteristics of the OMs of spirochetes and Gram-negative bacteria, it is also unique and different in ways that make it of general interest to microbiologists. For example, unlike most other pathogenic spirochetes, the leptospiral OM is rich in lipopolysaccharide (LPS). Leptospiral LPS is similar to that of Gram-negative bacteria but has a number of unique structural features that may explain why it is not recognized by the LPS-specific Toll-like receptor 4 of humans. As in other spirochetes, lipoproteins are major components of the leptospiral OM, though their roles are poorly understood. The functions of transmembrane outer membrane proteins (OMPs) in many cases are better understood, thanks to homologies with their Gram-negative counterparts and the emergence of improved genetic techniques. This chapter will review recent discoveries involving the leptospiral OM and its role in leptospiral physiology and pathogenesis.

Secretion of bacterial lipoproteins: through the cytoplasmic membrane, the periplasm and beyond.

Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., gram-positive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporter-like LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the "+2 rule". Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation, likely through interaction with a periplasmic holding chaperone, which delivers the proteins to an outer membrane lipoprotein flippase. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

A call to order at the spirochaetal host-pathogen interface.

As the Lyme disease spirochaete Borrelia burgdorferi shuttles back and forth between arthropod vector and vertebrate host, it encounters vastly different and hostile environments. Major mechanisms contributing to the success of this pathogen throughout this complex transmission cycle are phase and antigenic variation of abundant and serotype-defining surface lipoproteins. These peripherally membrane-anchored virulence factors mediate niche-specific interactions with vector/host factors and protect the spirochaete from the perils of the mammalian immune response. In this issue of Molecular Microbiology, Tilly, Bestor and Rosa redefine the roles of two lipoproteins, OspC and VlsE, during mammalian infection. Using a variety of promoter fusions in combination with a sensitive in vivo 'use it or lose it' gene complementation assay, the authors demonstrate that proper sequential expression of OspC followed by VlsE indeed matters. A previously suggested general functional redundancy between these and other lipoproteins is shown to be limited and dependent on an immunodeficient experimental setting that is arguably of diminished ecological relevance. These data reinforce the notion that OspC plays a unique role during initial infection while the antigenically variant VlsE proteins allow for persistence in the mammalian host.

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 plays 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.

Solid-phase polarization matrixes for dynamic nuclear polarization from homogeneously distributed radicals in mesostructured hybrid silica materials.

Mesoporous hybrid silica-organic materials containing homogeneously distributed stable mono- or dinitroxide radicals covalently bound to the silica surface were developed as polarization matrixes for solid-state dynamic nuclear polarization (DNP) NMR experiments. For TEMPO-containing materials impregnated with water or 1,1,2,2-tetrachloroethane, enhancement factors of up to 36 were obtained at ∼100 K and 9.4 T without the need for a glass-forming additive. We show that the homogeneous radical distribution and the subtle balance between the concentration of radical in the material and the fraction of radicals at a sufficient inter-radical distance to promote the cross-effect are the main determinants for the DNP enhancements we obtain. The material, as well as an analogue containing the poorly soluble biradical bTUrea, is used as a polarizing matrix for DNP NMR experiments of solutions containing alanine and pyruvic acid. The analyte is separated from the polarization matrix by simple filtration.

Molecular-level characterization of the structure and the surface chemistry of periodic mesoporous organosilicates using DNP-surface enhanced NMR spectroscopy.

We present the molecular level characterization of a phenylpyridine-based periodic mesoporous organosilicate and its post-functionalized organometallic derivatives through the fast acquisition of high quality natural isotopic abundance 1D (13)C, (15)N, and (29)Si and 2D (1)H-(13)C and (1)H-(29)Si solid-state NMR spectra enhanced with dynamic nuclear polarization.

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.

BpaB and EbfC DNA-binding proteins regulate production of the Lyme disease spirochete's infection-associated Erp surface proteins.

Vector-borne pathogens regulate their protein expression profiles, producing factors during host infection that differ from those produced during vector colonization. The Lyme disease agent, Borrelia burgdorferi, produces Erp surface proteins throughout mammalian infection and represses their synthesis during colonization of vector ticks. Known functions of Erp proteins include binding of host laminin, plasmin(ogen), and regulators of complement activation. A DNA region immediately 5' of erp operons, the erp operator, is required for transcriptional regulation. The B. burgdorferi BpaB and EbfC proteins exhibit high in vitro affinities for erp operator DNA. In the present studies, chromatin immunoprecipitation (ChIP) demonstrated that both proteins bind erp operator DNA in vivo. Additionally, a combination of in vivo and in vitro methods demonstrated that BpaB functions as a repressor of erp transcription, while EbfC functions as an antirepressor.

Borrelia burgdorferi, the Lyme disease spirochete, possesses genetically-encoded responses to doxycycline, but not to amoxicillin.

Some species of bacteria respond to antibiotic stresses by altering their transcription profiles, in order to produce proteins that provide protection against the antibiotic. Understanding these compensatory mechanisms allows for informed treatment strategies, and could lead to the development of improved therapeutics. To this end, studies were performed to determine whether Borrelia burgdorferi, the spirochetal agent of Lyme disease, also exhibits genetically-encoded responses to the commonly prescribed antibiotics doxycycline and amoxicillin. After culturing for 24 h in a sublethal concentration of doxycycline, there were significant increases in a substantial number of transcripts for proteins that are involved with translation. In contrast, incubation with a sublethal concentration of amoxicillin did not lead to significant changes in levels of any bacterial transcript. We conclude that B. burgdorferi has a mechanism(s) that detects translational inhibition by doxycycline, and increases production of mRNAs for proteins involved with translation machinery in an attempt to compensate for that stress.

Probing the Borrelia burgdorferi surface lipoprotein secretion pathway using a conditionally folding protein domain.

Surface lipoproteins of Borrelia spirochetes are important virulence determinants in the transmission and pathogenesis of Lyme disease and relapsing fever. To further define the conformational secretion requirements and to identify potential lipoprotein translocation intermediates associated with the bacterial outer membrane (OM), we generated constructs in which Borrelia burgdorferi outer surface lipoprotein A (OspA) was fused to calmodulin (CaM), a conserved eukaryotic protein undergoing calcium-dependent folding. Protein localization assays showed that constructs in which CaM was fused to full-length wild-type (wt) OspA or to an intact OspA N-terminal "tether" peptide retained their competence for OM translocation even in the presence of calcium. In contrast, constructs in which CaM was fused to truncated or mutant OspA N-terminal tether peptides were targeted to the periplasmic leaflet of the OM in the presence of calcium but could be flipped to the bacterial surface upon calcium chelation. This indicated that in the absence of an intact tether peptide, unfolding of the CaM moiety was required in order to facilitate OM traversal. Together, these data further support a periplasmic tether peptide-mediated mechanism to prevent premature folding of B. burgdorferi surface lipoproteins. The specific shift in the OM topology of sequence-identical lipopeptides due to a single-variable change in environmental conditions also indicates that surface-bound Borrelia lipoproteins can localize transiently to the periplasmic leaflet of the OM.