Fig 1.
Organization of operons containing fapH in various members of Campylobacterota and the predicted lipoprotein signal peptides for the FapH homologs.
(A) In many genomes, fapH is located immediately downstream of flgP. Approximate locations of predicted promoters for the operon containing fapH in H. pylori B128 are indicated (green arrows), which is based on a transcriptional start site database determined from the H. pylori 26695 transcriptome [63]. P-p: primary promoter, P-s: secondary promoter, P-i: internal promoter. (B) Predicted lipoprotein signal peptides of the FapH homologs. Amino acids sequences of the FapH homologs were analyzed for signal peptides and their cleavage sites using the SignalP-6.0 server (https://services.healthtech.dtu.dk/service.php?SignalP) [64]. Locations of the cleavage sites in the FapH homologs are indicated by the arrow.
Fig 2.
Electron micrographs of H. pylori B128 wild type and ΔfapH pflAT strain.
TEM images of representative cells of H. pylori B128 wild type (A) and ΔfapH pflAT mutant (B). Section of cryo-ET images of H. pylori B128 wild type (C) and ΔfapH pflAT mutant cells (D). In panels C and D, flagellar basal bodies are indicated by red arrows, flagellar sheath is indicated by the blue arrows, and flagellar filaments are indicated by the green arrows. OM–outer membrane; IM–inner membrane.
Fig 3.
Motility of H. pylori B128 wild type and ΔfapH mutants and their sensitivity to bacitracin.
(A) Motilities of wild type, ΔfapH pflAT, ΔfapH pflA*,ΔfapH pflAT/ppflA, and ΔfapH pflA*/pfapH were assessed by stab inoculating the strains in soft agar medium and then measuring the diameters of the resulting swim halos following a 7-d incubation period. Bars indicate mean values for the diameters of the swim halos and the error bars indicate the standard error of the mean (SEM). At least 4 replicates were used in motility assays. (B) Efficiency of plating assays were done with H. pylori strains (indicated in A) on TSA (-bac) or TSA supplemented with 200 μg/ml bacitracin (+bac). Cells from freshly grown cultures of the strains were resuspended in tryptic soy broth to the same cell densities (OD600). Ten-fold serial dilutions of the resuspensions (100 to 10−5) were then spotted onto the media, and the cultures were incubated for 7 d. The ΔpflA mutant was included to demonstrate that disrupting pflA was not responsible for the increased sensitivity to bacitracin. (C) Growth of H. pylori strains in soft agar medium in the absence (-bac) and presence of 200 μg/ml bacitracin (+bac). (D) H. pylori B128 wild type, ΔfapH pflA*, and ΔfapH pflA*/pfapH were stab inoculated into soft agar medium that contained 0, 100, or 200 μg/ml bacitracin and the diameters of the resulting swim halos were measured following a 7-d incubation period. Bars indicate mean values for swim halo diameters and the error bars indicate the SEM. The asterisk indicates statistically significant differences in swim halo diameters as determined using a two-sample t test (p-value <0.0001). At least 3 replicates were done for each sample.
Fig 4.
Architecture of the FapH ring in the H. pylori motor.
(A and B) In-situ structures of the flagellar motors from ΔfapH pflA* and wild-type cells were determined using subtomogram averaging, respectively. Medial slices through the motor structures indicate the ΔfapH pflA* motor is composed of all motor components, except globular densities in the outer membrane region. (C) A model showing the location of the FapH ring and outer disk structures. (D) Cross section through the globular densities in the ΔfapH pflA* motor. (E) Cross section through the globular densities in the wild-type motor. (F) 3D model of the FapH ring adjacent to the outer membrane. FR–FapH ring, OD–outer disk, BD–basal disk, OM–outer membrane, IM–inner membrane.
Fig 5.
Identification of FapH interaction partners.
(A) SDS-PAGE gel of proteins isolated from co-IP assay using anti-myc antibody. Lane 1 contained the molecular weight standards. Sizes of the protein standards are indicated to the left of the gel. Lanes 2 and 3 are negative controls and are samples from the co-IP assay with cellular extracts prepared from wild type H. pylori B128 and correspond to 5 μl and 10 μl of loaded sample, respectively. Lanes 3 and 4 are samples from the co-IP assay with cellular extracts prepared H. pylori B128 strain expressing the FapH-myc fusion protein and correspond to 5 μl and 10 μl loaded sample, respectively. Lane 6 shows a c-myc tagged E. coli protein (indicated by **) that served as a positive control for the co-IP assay and was supplied with the Pierce c-Myc Tag Magnetic IP/co-IP kit. Protein bands in lane 5 that were excised and analyzed to identify proteins present are indicated by an asterisk (*). HopD and FtsH were identified in the top band, the middle band corresponded to the FapH-myc protein, and HP1456 was identified in the lower band. (B) Results from β-galactosidase assays for strains with select combinations of adenylate cyclase T15 and T25 fragments fused to FapH, FlgP, HP1454, and HP1456. The cartoons shown on the y-axis illustrate the orientation of the adenylate cyclase fragments and H. pylori proteins in the fusion proteins relative to each other. In the cartoons, FapH is indicated in blue, FlgP is indicated in yellow, HP1456 is indicated in red, and HP1454 is indicated in green. pKNT25 and pKT25 indicate the adenylate cyclase T25 fragment at the N-terminus or C-terminus of the fusion protein, respectively. pUT18 and pUT18C indicate the adenylate cyclase T18 fragment at the N-terminus or C-terminus of the fusion protein, respectively. An E. coli strain containing the BACTH vectors pKT25 and pUT18 served as a negative control, and an E. coli strain bearing plasmids that expressed the T25-zip andT18-zip proteins served as a positive control. The bars indicate the average β-galactosidase activity for each strain and the error bars indicate the SEM. At least 3 replicates were done for each sample. A single asterisk (*) indicates a p-value of < 0.0001, two asterisks (**) indicate a p-value of < 0.001, and three asterisks (***) indicated a p-value of < 0.005 as determined using a two-sample t test. Significance of interaction between pKNT25-FlgP and FlgP-pUT18C is compared with pKT25 and pUT18. For the FapH-FlgP interactions, 7 of the 8 possible plasmid combinations produced significantly elevated levels of β-galactosidase activity, but only 3 of the 7 strains are shown in the figure. (C) Protein-protein interaction map constructed from co-IP and BACTH experiments. Solid black lines indicate interactions observed in co-IP assays involving the FapH-myc protein and dashed red lines indicate interactions observed in the BACTH assay.