Steven J. Norris

Antigenic variation in Lyme disease borreliae by promiscuous recombination of VMP-like sequence cassettes.

Abstract We have identified and characterized an elaborate genetic system in the Lyme disease spirochete Borrelia burgdorferi that promotes extensive antigenic variation of a surface-exposed lipoprotein, VlsE. A 28 kb linear plasmid of B. burgdorferi B31 (lp28–1) was found to contain a vmp -like sequence ( vls ) locus that closely resembles the variable major protein ( vmp ) system for antigenic variation of relapsing fever organisms. Portions of several of the 15 nonexpressed (silent) vls cassette sequences located upstream of vlsE recombined into the central vlsE cassette region during infection of C3H/HeN mice, resulting in antigenic variation of the expressed lipoprotein. This combinatorial variation could potentially produce millions of antigenic variants in the mammalian host.

A plasmid-encoded nicotinamidase (PncA) is essential for infectivity of Borrelia burgdorferi in a mammalian host

Borrelia burgdorferi, a spirochaete that causes Lyme borreliosis, contains 21 linear and circular plasmids thought to be important for survival in mammals or ticks. Our results demonstrate that the gene BBE22 encoding a nicotinamidase is capable of replacing the requirement for the 25 kb linear plasmid lp25 during mammalian infection. Transformation of B. burgdorferi lacking lp25 with a shuttle vector containing the lp25 gene BBE22 (pBBE22) restored infectivity in mice to a level comparable to that of wild‐type Borrelia. This complementation also restored the growth and host adaptation of lp25–B. burgdorferi in dialysis membrane chambers (DMCs) implanted in rats. A single Cys to Ala conversion at the putative active site of BBE22 abrogated the ability of pBBE22 to re‐establish infectivity or growth in DMCs. Additional Salmonella typhimurium complementation studies and enzymatic analysis demonstrated that the BBE22 gene product has nicotinamidase activity and is most probably required for the biosynthesis of NAD. These results indicate that some plasmid‐encoded products fulfil physiological functions required in the enzootic cycle of pathogenic Borrelia.

Disruption of the Genes Encoding Antigen 85A and Antigen 85B of Mycobacterium tuberculosis H37Rv: Effect on Growth in Culture and in Macrophages

ABSTRACT The mechanism of pathogenesis of Mycobacterium tuberculosis is thought to be multifactorial. Among the putative virulence factors is the antigen 85 (Ag85) complex. This family of exported fibronectin-binding proteins consists of members Ag85A, Ag85B, and Ag85C and is most prominently represented by 85A and 85B. These proteins have recently been shown to possess mycolyl transferase activity and likely play a role in cell wall synthesis. The purpose of this study was to generate strains of M. tuberculosis deficient in expression of the principal members of this complex in order to determine their role in the pathogenesis ofM. tuberculosis. Constructs of fbpA andfbpB disrupted with the kanamycin resistance marker ΩKm and containing varying amounts of flanking gene and plasmid vector sequences were then introduced as linear fragments into H37Rv by electroporation. Southern blot and PCR analyses revealed disruption of the homologous gene locus in one fbpA::ΩKm transformant and one fbpB::ΩKm transformant. The fbpA::ΩKm mutant, LAa1, resulted from a double-crossover integration event, whereas thefbpB::ΩKm variant, LAb1, was the product of a single-crossover type event that resulted in insertion of both ΩKm and plasmid sequences. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis confirmed that expression of the disrupted gene was not detectable in the fbpA andfbpB mutants. Analysis of growth rates demonstrated that the fbpB mutant LAb1 grew at a rate similar to that of the wild-type parent in enriched and nutrient-poor laboratory media as well as in human (THP-1) and mouse (J774.1A) macrophage-like cell lines. ThefbpA mutant LAa1 grew similarly to the parent H37Rv in enriched laboratory media but exhibited little or no growth in nutrient-poor media and macrophage-like cell lines. The targeted disruption of two genes encoding mycolyl transferase and fibronectin-binding activities in M. tuberculosis will permit the systematic determination of their roles in the physiology and pathogenesis of this organism.

BBE02 Disruption Mutants of Borrelia burgdorferi B31 Have a Highly Transformable, Infectious Phenotype

ABSTRACT We constructed highly transformable and infectious Borrelia burgdorferi B31 by inactivating BBE02, a putative restriction-modification gene on the linear plasmid lp25. The low-passage-number B31 clones 5A4 (containing all plasmids) and 5A18 (lp28-4− lp56−) were used for this study, and BBE02 was disrupted by homologous recombination. The transformation efficiency with the shuttle vector pBSV2C03::gntΔkan was increased from <1 to ∼10 colonies per μg of DNA for 5A4 and 5A4 BBE02::Kanr and from 14 to approximately 600 colonies per μg of DNA for 5A18 and 5A18 BBE02::Kanr. lp25, which is required for infectivity in mice, was retained in BBE02 mutants transformed with pBSV2C03::gntΔkan, but lp25 was not detected in transformants of the parental clones 5A4 and 5A18. BBE02 disruptants and pBSV2C03::gntΔkan transformants of these clones remained infectious in C3H/HeN mice, and the 50% infective doses of the BBE02 disruptants were <102 organisms per mouse. The inactivation of BBE02 thus eliminates a transformation barrier for infectious B. burgdorferi B31 and will provide a valuable tool for studying the virulence factors of Lyme disease.

Intact Flagellar Motor of Borrelia burgdorferi Revealed by Cryo-Electron Tomography: Evidence for Stator Ring Curvature and Rotor/C-Ring Assembly Flexion

The bacterial flagellar motor is a remarkable nanomachine that provides motility through flagellar rotation. Prior structural studies have revealed the stunning complexity of the purified rotor and C-ring assemblies from flagellar motors. In this study, we used high-throughput cryo-electron tomography and image analysis of intact Borrelia burgdorferi to produce a three-dimensional (3-D) model of the in situ flagellar motor without imposing rotational symmetry. Structural details of B. burgdorferi, including a layer of outer surface proteins, were clearly visible in the resulting 3-D reconstructions. By averaging the 3-D images of approximately 1,280 flagellar motors, a approximately 3.5-nm-resolution model of the stator and rotor structures was obtained. flgI transposon mutants lacked a torus-shaped structure attached to the flagellar rod, establishing the structural location of the spirochetal P ring. Treatment of intact organisms with the nonionic detergent NP-40 resulted in dissolution of the outermost portion of the motor structure and the C ring, providing insight into the in situ arrangement of the stator and rotor structures. Structural elements associated with the stator followed the curvature of the cytoplasmic membrane. The rotor and the C ring also exhibited angular flexion, resulting in a slight narrowing of both structures in the direction perpendicular to the cell axis. These results indicate an inherent flexibility in the rotor-stator interaction. The FliG switching and energizing component likely provides much of the flexibility needed to maintain the interaction between the curved stator and the relatively symmetrical rotor/C-ring assembly during flagellar rotation.

Decreased Electroporation Efficiency in Borrelia burgdorferi Containing Linear Plasmids lp25 and lp56: Impact on Transformation of Infectious B. burgdorferi

ABSTRACT The presence of the linear plasmids lp25 and lp56 of Borrelia burgdorferi B31 was found to dramatically decrease the rate of transformation by electroporation with the shuttle vector pBSV2, an autonomously replicating plasmid that confers kanamycin resistance (P. E. Stewart, R. Thalken, J. L. Bono, and P. Rosa, Mol. Microbiol. 39:714-721, 2001). B. burgdorferi B31 clones had transformation efficiencies that were either low, intermediate, or high, and this phenotype correlated with the presence or absence of lp25 and lp56. Under the conditions utilized in this study, no transformants were detected in clones that contained both lp25 and lp56; the few kanamycin-resistant colonies isolated did not contain pBSV2, indicating that the resistance was due to mutation. Intermediate electroporation rates (10 to 200 colonies per μg of DNA) were obtained with B31 clones that were either lp25− and lp56+ or lp25+ and lp56−. Clones in this group that initially contained lp25 lacked this plasmid in pBSV2 transformants, a finding consistent with selective transformation of lp25− variants. High transformation rates (>1,000 colonies per μg of DNA) occurred in clones that lacked both lp25 and lp56. Sequence analysis indicated that lp25 and lp56 contain genes that may encode restriction and/or modification systems that could result in the low transformation rates obtained with strains containing these plasmids. The previously reported correlation between lp25 and infectivity in mice, coupled with the barrier lp25 presents to transformation, may explain the difficulty in obtaining virulent transformants of B. burgdorferi.

Antigenic variation with a twist--the Borrelia story.

A common mechanism of immune evasion in pathogenic bacteria and protozoa is antigenic variation, in which genetic or epigenetic changes result in rapid, sequential shifts in a surface‐exposed antigen. In this issue of Molecular Microbiology, Dai et al. provide the most complete description to date of the vlp/vsp antigenic variation system of the relapsing fever spirochaete, Borrelia hermsii. This elaborate, plasmid‐encoded system involves an expression site that can acquire either variable large protein (vlp) or variable small protein (vsp) surface lipoprotein genes from 59 different archival copies. The archival vlp and vsp genes are arranged in clusters on at least five different plasmids. Gene conversion occurs through recombination events at upstream homology sequences (UHS) found in each gene copy, and at downstream homology sequences (DHS) found periodically among the vlp/vsp archival genes. Previous studies have shown that antigenic variation in relapsing fever Borrelia not only permits the evasion of host antibody responses, but can also result in changes in neurotropism and other pathogenic properties. The vlsE antigenic variation locus of Lyme disease spirochaetes, although similar in sequence to the relapsing fever vlp genes, has evolved a completely different antigenic variation mechanism involving segmental recombination from a contiguous array of vls silent cassettes. These two systems thus appear to represent divergence from a common precursor followed by functional convergence to create two distinct antigenic variation processes.

Role of Acetyl-Phosphate in Activation of the Rrp2-RpoN-RpoS Pathway in Borrelia burgdorferi

Borrelia burgdorferi, the Lyme disease spirochete, dramatically alters its transcriptome and proteome as it cycles between the arthropod vector and mammalian host. During this enzootic cycle, a novel regulatory network, the Rrp2-RpoN-RpoS pathway (also known as the σ54–σS sigma factor cascade), plays a central role in modulating the differential expression of more than 10% of all B. burgdorferi genes, including the major virulence genes ospA and ospC. However, the mechanism(s) by which the upstream activator and response regulator Rrp2 is activated remains unclear. Here, we show that none of the histidine kinases present in the B. burgdorferi genome are required for the activation of Rrp2. Instead, we present biochemical and genetic evidence that supports the hypothesis that activation of the Rrp2-RpoN-RpoS pathway occurs via the small, high-energy, phosphoryl-donor acetyl phosphate (acetyl∼P), the intermediate of the Ack-Pta (acetate kinase-phosphate acetyltransferase) pathway that converts acetate to acetyl-CoA. Supplementation of the growth medium with acetate induced activation of the Rrp2-RpoN-RpoS pathway in a dose-dependent manner. Conversely, the overexpression of Pta virtually abolished acetate-induced activation of this pathway, suggesting that acetate works through acetyl∼P. Overexpression of Pta also greatly inhibited temperature and cell density-induced activation of RpoS and OspC, suggesting that these environmental cues affect the Rrp2-RpoN-RpoS pathway by influencing acetyl∼P. Finally, overexpression of Pta partially reduced infectivity of B. burgdorferi in mice. Taken together, these findings suggest that acetyl∼P is one of the key activating molecule for the activation of the Rrp2-RpoN-RpoS pathway and support the emerging concept that acetyl∼P can serve as a global signal in bacterial pathogenesis.

Detailed analysis of sequence changes occurring during vlsE antigenic variation in the mouse model of Borrelia burgdorferi infection.

Lyme disease Borrelia can infect humans and animals for months to years, despite the presence of an active host immune response. The vls antigenic variation system, which expresses the surface-exposed lipoprotein VlsE, plays a major role in B. burgdorferi immune evasion. Gene conversion between vls silent cassettes and the vlsE expression site occurs at high frequency during mammalian infection, resulting in sequence variation in the VlsE product. In this study, we examined vlsE sequence variation in B. burgdorferi B31 during mouse infection by analyzing 1,399 clones isolated from bladder, heart, joint, ear, and skin tissues of mice infected for 4 to 365 days. The median number of codon changes increased progressively in C3H/HeN mice from 4 to 28 days post infection, and no clones retained the parental vlsE sequence at 28 days. In contrast, the decrease in the number of clones with the parental vlsE sequence and the increase in the number of sequence changes occurred more gradually in severe combined immunodeficiency (SCID) mice. Clones containing a stop codon were isolated, indicating that continuous expression of full-length VlsE is not required for survival in vivo; also, these clones continued to undergo vlsE recombination. Analysis of clones with apparent single recombination events indicated that recombinations into vlsE are nonselective with regard to the silent cassette utilized, as well as the length and location of the recombination event. Sequence changes as small as one base pair were common. Fifteen percent of recovered vlsE variants contained “template-independent” sequence changes, which clustered in the variable regions of vlsE. We hypothesize that the increased frequency and complexity of vlsE sequence changes observed in clones recovered from immunocompetent mice (as compared with SCID mice) is due to rapid clearance of relatively invariant clones by variable region-specific anti-VlsE antibody responses.

Identification and transcriptional analysis of a Treponema pallidum operon encoding a putative ABC transport system, an iron-activated repressor protein homolog, and a glycolytic pathway enzyme homolog

We have characterized a 5.2-kilobase (kb) putative transport related operon (tro) locus of Treponema pallidum subsp. pallidum (Nichols strain) (Tp) encoding six proteins: TroA, TroB, TroC, TroD, TroR and Phosphoglycerate mutase (Pgm). Four of these gene products (TroA-TroD) are homologous to members of the ATP-Binding Cassette (ABC) superfamily of bacterial transport proteins. TroA (previously identified as Tromp1) has significant sequence similarity to a family of Gram-negative periplasmic substrate-binding proteins and to a family of streptococcal proteins that may have dual roles as substrate binding proteins and adhesins. TroB is homologous to the ATP-binding protein component, whereas TroC and TroD are related to the hydrophobic membrane protein components of ABC transport systems. TroR is similar to Gram-positive iron-activated repressor proteins (DesR, DtxR, IdeR, and SirR). The last open reading frame (ORF) of the tro operon encodes a protein that is highly homologous to the glycolytic pathway enzyme, Pgm. Primer extension results demonstrated that the tro operon is transcribed from a sigma 70-type promoter element. Northern analysis and reverse transcriptase-polymerase chain reactions provided evidence for the presence of a primary 1-kb troA transcript and a secondary, less abundant, troA-pgm transcript. The tro operon is flanked by a Holliday structure DNA helicase homolog (upstream) and two ORFs representing a purine nucleoside phosphorylase homolog and tpp15, a previously characterized gene encoding a membrane lipoprotein (downstream). The presence of a complex operon containing a putative ABC transport system and a DtxR homolog indicates a possible linkage between transport and gene regulation in Tp.