Cryo-electron tomography of periplasmic flagella in Borrelia burgdorferi reveals a distinct cytoplasmic ATPase complex.

Periplasmic flagella are essential for the distinct morphology and motility of spirochetes. A flagella-specific type III secretion system (fT3SS) composed of a membrane-bound export apparatus and a cytosolic ATPase complex is responsible for the assembly of the periplasmic flagella. Here, we deployed cryo-electron tomography (cryo-ET) to visualize the fT3SS machine in the Lyme disease spirochete Borrelia burgdorferi. We show, for the first time, that the cytosolic ATPase complex is attached to the flagellar C-ring through multiple spokes to form the “spoke and hub� structure in B. burgdorferi. This structure not only strengthens structural rigidity of the round-shaped C-ring but also appears to rotate with the C-ring. Our studies provide structural insights into the unique mechanisms underlying assembly and rotation of the periplasmic flagella and may provide the basis for the development of novel therapeutic strategies against several pathogenic spirochetes

Traditional Characteristics of Treponemal Infections

<p>Traditional Characteristics of Treponemal Infections</p

A Phylogenetic Tree Depicting the Relationships between <i>T. pallidum</i> strains.

<p>This phylogeny is identical to the phylogeny of Harper et al. (Fig. 2) except that it has been re-estimated to show only branches with >50% bootstrap support for both maximum likelihood and maximum parsimony analyses. As with Harper et al.'s phylogeny, vertical distance is not meaningful, i.e. the placement of taxa along the vertical lines is inconsequential. <i>Pertenue</i>, <i>endemicum</i> and <i>pallidum</i> strains are depicted in green, blue, and red, respectively.</p

Comparison of heterogeneous positions identified in TPA SS14 strain by Matějková et al. [14] and by the automated pipeline used in this study.

<p>^aadditional intrastrain heterogeneous genome positions identified by Matějková et al. [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004110#pntd.0004110.ref014" target="_blank">14</a>] including 135141, 135144, 135149, 135220, 135227, 671982, 672004, 672016, 672025, 672026, 672027, 672028, 672036, 672039, 672040, 672041, 672042, 672043, 672044, 672154, 673088, 673119, 673511, 673545, 673550, and 673554 (according to the CP000805.1) were located in paralogous regions and therefore were excluded from the automated pipeline (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004110#pntd.0004110.s002" target="_blank">S2 Table</a>)</p><p>^bnumbers in parentheses show numbers of sequenced clones [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004110#pntd.0004110.ref014" target="_blank">14</a>] or nucleotide frequency within individual Illumina sequence reads (this study); NA—not available</p><p>^cnot present in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004110#pntd.0004110.t002" target="_blank">Table 2</a>; heterogeneous positions were detected in raw Illumina sequencing reads but were excluded due to study criteria</p><p>^d these heterogeneous sites were not found among Illumina reads, but were identified among 454 reads (SRX000109)</p><p>^esee also <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004110#pntd.0004110.t003" target="_blank">Table 3</a>; independent DNA preparations showed clear differences in proportions of alternative alleles, ranging from 0.1 to 0.7</p><p>Comparison of heterogeneous positions identified in TPA SS14 strain by Matějková et al. [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004110#pntd.0004110.ref014" target="_blank">14</a>] and by the automated pipeline used in this study.</p

Data analysis workflow.

<p><b>(A)</b> An automated identification pipeline and optimization process. <b>(B)</b> An application of further restrictions and verification of identified putative candidates.</p

Summary of the intrastrain variable sites identified within Illumina sequencing reads in investigated treponemal genomes.

<p>Illumina-identified intrastrain variable sites were verified using 454 or Sanger sequencing.</p><p>^ano intrastrain heterogeneous site were identified in the TPA Mexico A, TPE CDC-2, TPE Gauthier, TPE Fribourg-Blanc and TPLC Cuniculi A genomes</p><p>^bnonconservative amino acid replacements are not listed</p><p>^cif not indicated, localization was predicted by PSORTb</p><p>^dnot applicable</p><p>^e[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004110#pntd.0004110.ref020" target="_blank">20</a>],[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004110#pntd.0004110.ref023" target="_blank">23</a>]</p><p>^fvariable number of direct repeat (TCCTCCCCC)</p><p>Summary of the intrastrain variable sites identified within Illumina sequencing reads in investigated treponemal genomes.</p

Analysis of an Ordered, Comprehensive STM Mutant Library in Infectious <em>Borrelia burgdorferi</em>: Insights into the Genes Required for Mouse Infectivity

<div><p>The identification of genes important in the pathogenesis of Lyme disease <em>Borrelia</em> has been hampered by exceedingly low transformation rates in low-passage, infectious organisms. Using the infectious, moderately transformable <em>B. burgdorferi</em> derivative 5A18NP1 and signature-tagged versions of the <em>Himar1</em> transposon vector pGKT, we have constructed a defined transposon library for the efficient genome-wide investigation of genes required for wild-type pathogenesis, in vitro growth, physiology, morphology, and plasmid replication. To facilitate analysis, the insertion sites of 4,479 transposon mutants were determined by sequencing. The transposon insertions were widely distributed across the entire <em>B. burgdorferi</em> genome, with an average of 2.68 unique insertion sites per kb DNA. The 10 linear plasmids and 9 circular plasmids had insertions in 33 to 100 percent of their predicted genes. In contrast, only 35% of genes in the 910 kb linear chromosome had incapacitating insertions; therefore, the remaining 601 chromosomal genes may represent essential gene candidates. In initial signature-tagged mutagenesis (STM) analyses, 434 mutants were examined at multiple tissue sites for infectivity in mice using a semi-quantitative, Luminex-based DNA detection method. Examples of genes found to be important in mouse infectivity included those involved in motility, chemotaxis, the phosphoenolpyruvate phosphotransferase system, and other transporters, as well as putative plasmid maintenance genes. Availability of this ordered STM library and a high-throughput screening method is expected to lead to efficient assessment of the roles of <em>B. burgdorferi</em> genes in the infectious cycle and pathogenesis of Lyme disease.</p> </div

Maps of the STM transposon insertion points in representative plasmids of <i>B. burgdorferi</i> B31 5A18NP1.

<p>The following plasmids are shown: cp26 (Plasmid B, 26,498 bp, 307 transposon mutants, 250 unique sites), lp25 (Plasmid E, 24,177 bp, 102 transposon mutants, 85 unique sites), lp28-1 (Plasmid F, 26,921 bp, 83 transposon mutants, 71 unique sites), lp36 (Plasmid K, 29,766 bp, 224 transposon mutants, 209 unique sites), and lp54 (Plasmid A, 223 transposon mutants, 195 unique sites). Transposon insertion sites are depicted as blue triangles. ORFs that represent apparent pseudogenes or gene fragments are indicated by asterisks after the gene number. Regions that lack transposon insertions are indicated by orange rectangles below each map. A key for the color coding of paralogous gene families commonly associated with the origins of replication of <i>B. burgdorferi</i> plasmids is provided at the bottom of the figure. Maps of the transposon insertion sites for all of the plasmids are provided in Supporting Information, Fig. 3 in File S1.</p

Effects of transposon mutations in genes involved in flagellar assembly and chemotaxis on <i>I. scapularis</i> nymph transmission of <i>B. burgdorferi</i> clones to C3H/HeN mice.^a

<p> <b>Results obtained by needle inoculation and Luminex STM analysis are shown for comparison.^b</b></p><p>a Ticks were infected by capillary feeding with <i>in vitro</i> cultured <i>B. burgdorferi</i> and then fed on uninfected mice (see Materials and Methods). Separate groups of ticks were infected with each <i>B. burgdorferi</i> clone and used for tick inoculation of groups of 3 mice. The parental strain B315A18NP1 was used as a positive control in each of the 6 tick inoculation experiments shown.</p><p>b For comparison, needle inoculation STM results for the same clones are shown; these represent the cumulative results for all time points and tissues (see Spreadsheet S2). Results obtained for the STM positive control strain T02P01A01 in the corresponding STM experiments are provided at the bottom of the table.</p><p>c Number of spirochetes per field as detected by direct immunofluorescence with an anti-<i>B. burgdorferi</i> antiserum.</p><p>d Antibody to the VlsE C6 peptide.</p><p>e ND  =  Not Done.</p><p>f Culture positivity was due to the presence of a second clone with an unaltered <i>cheY-2</i> gene in the T05TC230 culture.</p><p>gMG064 is a transposon mutant from a non-STM library (D. J. Botkin and S. J. Norris, unpublished data). Chromosomal insertion site  = 721,311 bp.</p

Transposon mutations in Protein Family (PF) genes postulated to be involved in plasmid maintenance.

<p>PF gene families are listed across the top. Presence of a full-length PF gene of each family is indicated by a colored circle. The plasmid and plasmid-associated PF genes are indicated on the left and right sides, respectively; the PF genes are listed in the same order (left to right) as in the figure. lp28-1 has two PF gene loci. Genes with multiple transposon insertions are marked as XX, whereas those with single mutations have a single X. Genes that have insertions only at the end of the gene are marked by an asterisk; the adjacent number indicates the insertion ratio.</p