Loss of a Cardiolipin Synthase in Helicobacter pylori G27 Blocks Flagellum Assembly

Abstract

Helicobacter pylori uses a cluster of polar, sheathed flagella for motility, which it requires for colonization of the gastric epithelium in humans. As part of a study to identify factors that contribute to localization of the flagella to the cell pole, we disrupted a gene encoding a cardiolipin synthase (clsC) in H. pylori strains G27 and B128. Flagellum biosynthesis was abolished in the H. pylori G27 clsC mutant but not in the B128 clsC mutant. Transcriptome sequencing analysis showed that flagellar genes encoding proteins needed early in flagellum assembly were expressed at wild-type levels in the G27 clsC mutant. Examination of the G27 clsC mutant by cryo-electron tomography indicated the mutant assembled nascent flagella that contained the MS ring, C ring, flagellar protein export apparatus, and proximal rod. Motile variants of the G27 clsC mutant were isolated after allelic exchange mutagenesis using genomic DNA from the B128 clsC mutant as the donor. Genome resequencing of seven motile G27 clsC recipients revealed that each isolate contained the flgI (encodes the P-ring protein) allele from B128. Replacing the flgI allele in the G27 clsC mutant with the B128 flgI allele rescued flagellum biosynthesis. We postulate that H. pylori G27 FlgI fails to form the P ring when cardiolipin levels in the cell envelope are low, which blocks flagellum assembly at this point. In contrast, H. pylori B128 FlgI can form the P ring when cardiolipin levels are low and allows for the biosynthesis of mature flagella.IMPORTANCEH. pylori colonizes the epithelial layer of the human stomach, where it can cause a variety of diseases, including chronic gastritis, peptic ulcer disease, and gastric cancer. To colonize the stomach, H. pylori must penetrate the viscous mucous layer lining the stomach, which it accomplishes using its flagella. The significance of our research is identifying factors that affect the biosynthesis and assembly of the H. pylori flagellum, which will contribute to our understanding of motility in H. pylori, as well as other bacterial pathogens that use their flagella for host colonization.

Keywords: Helicobacter pylori; cardiolipin; cardiolipin synthase; flagellum.

FIG 1

Effects of clsC knockouts on motility and number of flagella per cell in H. pylori G27 and B128. (A) Motility was assessed on soft agar medium. Measurements indicate the halo diameters surrounding the point of inoculation after 7 days. Asterisks (single for G27 and double for B128) indicate values that were significantly different (P < 0.01) from that of the parental strain. Statistical significance was determined using the two-sample t test. (B and C) The numbers of flagella per cell were determined by TEM after negative staining (n = 100 per strain). The asterisk indicates a value significantly different from that of the parental strain (P < 0.05). Statistical significance was determined using the Mann-Whitney U test.

FIG 2

ClsC from H. pylori strains G27 and B128 is involved in CL synthesis. Radiolabeled glycerophospholipids from E. coli W3310 (lane 1; standard) and various H. pylori strains (lanes 2 through 9) were isolated and separated by TLC. Glycerophospholipids were separated in the following order (bottom to top): phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL). Wild-type strains of H. pylori G27 and B128 (lanes 2 and 6) displayed similar patterns for glycerophospholipid species with two unique spots for CL. The CL-deficient strains (lanes 3 and 7) show a loss of the top CL spot in both strains. Introducing the complementing plasmid (pCls), but not the empty shuttle vector (pHel3; lanes 4 and 8), in both mutants (lanes 5 and 9) recovers CL levels, indicated by the presence of the top CL spot for these strains.

FIG 3

MALDI-TOF mass spectrometry of clsC mutants in H. pylori G27 and B128. (A) The wild-type and complemented strains produced peaks in the CL ranges of 1,150 and 1,400 m/z as reported for H. pylori (36). Samples from the wild-type and complemented strains displayed peaks with m/z values of 1,189.9, 1,261.8, 1,329.9, and 1,397.9. In contrast, the sample from the B128 clsC mutant only displayed a peak with an m/z value of 1,189.9, while the G27 clsC mutant showed peaks with m/z values of 1,189.9 and 1,397.9. (B) Predicted structures for the CL species corresponding to the peaks from the mass spectrometry analysis. MALDI-TOF was performed in the negative mode, resulting in negatively charged ions. The error of the instrument is ±1, and so the observed m/z values are consistent with the calculated masses for the Na adducts.

FIG 4

Genes identified in RNA-seq to be differentially regulated. Each bar represents a gene with a log₂ fold change of ±1.5 in the G27 clsC mutant. Functions for the genes indicated are as follows: flgE, flagellar hook protein; flaB, minor flagellin B; crdA, copper resistance determinant; flgJ, predicted muramidase; flgK, flagellar hook-associated protein; flgL, flagellar hook-associated protein; sabA, outer membrane protein; and cagB, cag pathogenicity island protein B. The remaining genes either encode proteins of unknown function or are pseudogenes.

FIG 5

Expression levels of select flagellar genes as determined by RT-qPCR. Each bar indicates the mean fold change for three biological replicates in a comparison of transcript levels of the target genes from the G27 clsC and the G27 wild-type strain. Fold change values for all target genes were calculated using the ΔΔC_T method (63) using rpoA and rpoD as reference genes. A statistically significant difference between the mutant and wild-type strains for the middle and late genes was observed using the two-sample t test (P < 0.01).

FIG 6

In situ structure of the nascent flagellum from the H. pylori G27 clsC mutant. (A) Representative slice of a three-dimensional reconstruction of H. pylori G27 clsC. (B) Zoom-in view showing the MS/C-ring together with the partial rod density. (C) Central section slice of a subtomogram-averaged structure from H. pylori G27 clsC. (D) Central section slice from wild-type motor-averaged structure. The scale bars in panels A and B are 50 nm in length, while the scale bars in panels C and D are 20 nm in length. OM, outer membrane; IM, inner membrane.

FIG 7

Motility and numbers of flagella per cell for the G27 clsC motile variants isolated after allelic exchange mutagenesis. (A) Motility was assessed on soft agar medium. Measurements indicate the halo diameters surrounding the point of inoculation after 7 days. The statistical significance was determined using the two-sample t test. (B) The number of flagella per cell was assessed by TEM after negative staining (n = 100 per strain). The statistical significance was determined using a Mann-Whitney U test. For both panels, one asterisk indicates that a strain is statistically different compared to the H. pylori G27 clsC parental strain (P < 0.01) and two asterisks indicate that a strain is statistically different compared to the motile variant mv49 (P < 0.01).

FIG 8

Motility and flagellum count results for the G27 clsC mutant strains in which specific B128 alleles were introduced. (A) Motility was assessed on soft agar medium. Measurements indicate the halo size surrounding the point of inoculation after 7 days. The G27 wild-type and G27 clsC strains are used as references. Three isolates of the G27 clsC mutant in which the B128 icfA and kdsA alleles (iso146, iso147, and iso148) or the B128 flgI allele (iso164, iso168, and iso169) had been introduced were examined in the motility assay. Statistical significance was determined by using a two-sample t test. (B) The number of flagella per cell was assessed by TEM after negative staining (n = 100 per strain).The statistical significance was determined by using a Mann-Whitney U test. The asterisks indicate a statistically significant difference in the number of flagella per cell for the G27 clsC mutants bearing the B128 flgI allele (iso164, iso168, and iso169) compared to the G27 clsC parental strain (P < 0.01).