Novel components of the flagellar system in epsilonproteobacteria

Abstract

Motility is essential for the pathogenesis of many bacterial species. Most bacteria move using flagella, which are multiprotein filaments that rotate propelled by a cell wall-anchored motor using chemical energy. Although some components of the flagellar apparatus are common to many bacterial species, recent studies have shown significant differences in the flagellar structures of different bacterial species. The molecular bases for these differences, however, are not understood. The flagella from epsilonproteobacteria, which include the bacterial pathogens Campylobacter jejuni and Helicobacter pylori, are among the most divergent. Using next-generation sequencing combined with transposon mutagenesis, we have conducted a comprehensive high-throughput genetic screen in Campylobacter jejuni, which identified several novel components of its flagellar system. Biochemical analyses detected interactions between the identified proteins and known components of the flagellar machinery, and in vivo imaging located them to the bacterial poles, where flagella assemble. Most of the identified new components are conserved within but restricted to epsilonproteobacteria. These studies provide insight into the divergent flagella of this group of bacteria and highlight the complexity of this remarkable structure, which has adapted to carry out its conserved functions in the context of widely diverse bacterial species.

Importance: Motility is essential for the normal physiology and pathogenesis of many bacterial species. Most bacteria move using flagella, which are multiprotein filaments that rotate propelled by a motor that uses chemical energy as fuel. Although some components of the flagellar apparatus are common to many bacterial species, recent studies have shown significant divergence in the flagellar structures across bacterial species. However, the molecular bases for these differences are not understood. The flagella from epsilonproteobacteria, which include the bacterial pathogens Campylobacter jejuni and Helicobacter pylori, are among the most divergent. We conducted a comprehensive genetic screen in Campylobacter jejuni and identified several novel components of the flagellar system. These studies provide important information to understand how flagella have adapted to function in the context of widely diverse sets of bacterial species and bring unique insight into the evolution and function of this remarkable bacterial organelle.

FIG 1

INSeq transposon mutagenesis of C. jejuni. (A) Schematic depiction of the transposable element used in this study. Arrows represent the direction of the promoters of the chloramphenicol acetyltransferase (Cat) and kanamycin (Km) antibiotic resistance genes. (B) C. jejuni 81-176 genome map depicting the distribution of transposon insertions in the mutant library. The outer (first) circle depicts sequence length (in bases); the second, the number of reads per kb of the genome (white, 0 reads; orange, ≤10; magenta, ≤100; red, >100); the third (blue), essential genes; the fourth (cyan), nonessential genes; and the fifth, GC skew (G−C/G+C) (khaki, values > 0; purple, values < 0). (C) Reproducibility of experimental protocols. Technical replicates were prepared and sequenced from a single transposon mutant population. Each point represents the abundance of read numbers of a single gene, which is normalized to 1 million reads. The coefficient of determination, R², value corresponding to the log-transformed abundance value is 0.97. (D) Relative abundances of insertion mutants before and after cultured mammalian cell infection. The relative abundances of mutations in each gene (points) in the input (before infection) and output (after infection) populations were compared. Genes that showed a statistically significant change (q <0.01) in representation in all 3 biological replicates are shown in red; the others are shown in black. The R² value corresponding to the log-transformed abundance value is 0.93.

FIG 2

Ability of the C. jejuni 81-176 mutants to invade cultured mammalian cells. Cultured mammalian cells were infected with the C. jejuni 81-176 wild type (WT), the indicated mutants, or the complemented mutant strains at an MOI of 100 for 2 h, followed by 2 h of incubation in the presence of gentamicin. Levels of invasion are shown as the percentages of bacteria that survived treatment with gentamicin relative to the WT, whose level was set at 100%. Values are means ± standard errors of results of 3 independent determinations. The difference between the value for the knockout mutants and that for the WT or the complemented mutant was statistically significant (P < 0.001).

FIG 3

Motility analysis of the invasion-defective C. jejuni mutants and complemented strains on soft agar. WT, wild-type C. jejuni; −, deletion mutant; +, complemented mutant of the specified gene; +FlhA, complementation of the indicated mutant with the flhA gene.

FIG 4

Transmission electron microscopy analysis of the nonmotile mutant strains of C. jejuni. WT, wild-type C. jejuni.

FIG 5

Subcellular localization of C. jejuni proteins involved in motility. C. jejuni strains expressing the indicated sfGFP-tagged proteins were examined by fluorescence microscopy. The quantification of the proportion of bacteria exhibiting GFP localization at one or both poles or showing no polar fluorescence is shown. CJJ81176_0100_GFP n = 101; CJJ81176_0413_GFP n = 285; CJJ81176_1488_GFP n = 327; sfGFP n = 87. sfGFP, superfolder green fluorescent protein; DIC, differential interference contrast.

FIG 6

Confirmation of protein interactions by Western immunoblot analysis. C. jejuni strains carrying M45-epitope-tagged or FLAG-epitope-tagged versions of the interacting proteins (as indicated) were subjected to immunoprecipitation with anti-FLAG affinity gels (or control beads), and the immunoprecipitated materials were analyzed by Western immunoblotting using an anti-M45 antibody.

FIG 7

Interaction map of the C. jejuni motility proteins. The red lines with arrowheads indicate interactions confirmed by coimmunoprecipitation. Other interactions are adopted from the STRING database 9.0 (http://string-db.org/) using the highest confidence (0.9) parameters. The analyzed C. jejuni proteins are indicated in red.

FIG 8

Schematic of the flagellar structure depicting the identified C. jejuni flagellar proteins and their interactions. OM, outer membrane; IM, inner membrane; PG, peptidoglycan.