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Article

Localization of a Cardiolipin Synthase in Helicobacter pylori and Its Impact on the Flagellar Sheath Proteome

by
Doreen Nguyen
1,
Nathan East
1,
Vincent J. Starai
1,2 and
Timothy R. Hoover
1,*
1
Department of Microbiology, University of Georgia, Athens, GA 30602, USA
2
Department of Infectious Diseases, University of Georgia, Athens, GA 30602, USA
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2025, 16(7), 155; https://doi.org/10.3390/microbiolres16070155
Submission received: 5 June 2025 / Revised: 24 June 2025 / Accepted: 1 July 2025 / Published: 7 July 2025
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Abstract

Helicobacter pylori, which colonizes the human gastric mucosa, uses a cluster of polar, sheathed flagella to swim across the mucous layer of the stomach. The function and biogenesis of the H. pylori flagellar sheath are poorly understood. Cardiolipin is a phospholipid that accumulates in regions of the membrane that have negative curvature, such as the cell pole, cell septum, and flagellar sheath. The final step in cardiolipin biosynthesis is catalyzed by cardiolipin synthase. H. pylori has at least two cardiolipin synthases, one of which is cardiolipin synthase C (ClsC). Bioinformatic analysis revealed that homologs of H. pylori ClsC are restricted to Helicobacter species that have sheathed flagella and the ClsC homologs are predicted lipoproteins. Fluorescence microscopy revealed that a ClsC super-folder green fluorescent protein localized to the cell pole and cell septum in H. pylori G27. Comparing the proteomes of isolated sheathed flagella from the H. pylori B128 wild type and a clsC::cat mutant, we identified five proteins that were absent in the mutant flagellum preparations. One of the proteins was FaaA, an autotransporter that localizes to the flagellar sheath. These findings suggest that the localization of FaaA and possibly other proteins to the flagellar sheath is dependent on ClsC.

1. Introduction

Helicobacter pylori is a Gram-negative, helical-shaped bacterium that belongs to the phylum Campylobacterota. H. pylori maintains a stable presence in the highly specific ecological niche of the human gastric mucosa, and it is estimated that approximately half of the world’s population is infected with the bacterium [1]. While the majority of individuals infected with H. pylori are asymptomatic, H. pylori infections can lead to a variety of gastric pathologies, including chronic gastritis, peptic ulcer disease, and gastric cancer [2,3,4]. Gastric cancer accounts for one-third of cancer-related deaths, and the majority of these gastric cancer-related deaths are attributed to H. pylori infection [2,5]. Due to the linkage between H. pylori infection and gastric cancer, H. pylori was labeled as a class I carcinogen in 1994 by the International Agency for Research on Cancer and the World Health Organization [6].
H. pylori cells typically possess four to six sheathed, polar flagella that are used for motility, which is required for the colonization of the gastric mucosa in animal models [7,8]. The flagellar sheath surrounds the filament and is contiguous with the outer membrane [9]. The flagellar sheath is a structure that is found in a number of other bacteria, including most Helicobacter and Vibrio species [10]. Although the role of the flagellar sheath in H. pylori is not known, proposed functions include the protection of the flagellar filament from depolymerization by gastric acid and the promotion of adherence to the gastric epithelium [9,11]. The flagellar sheaths of some bacterial species have been suggested to be important in avoiding the detection of the flagellins (i.e., flagellar filament proteins) by the host innate immune system [10]. Bacterial flagellins activate Toll-like receptor 5 (TLR5) in the host to induce interleukin-8 (IL-8) secretion as part of the innate immune response [12]. In support of this idea, Yoon and Mekalanos showed that, even though the flagellins of Vibrio cholerae and Salmonella enterica serovar Typhimurium (S. Typhimurium) elicit similar TLR5-mediated innate immune responses, the sheathed flagella of V. cholerae display a significant reduction in triggering a host innate immune response compared to the unsheathed S. Typhimurium flagella [13]. The H. pylori flagellar sheath likely does not play the primary role in avoiding surveillance by the innate immune system, however, as H. pylori flagellins are significantly less potent than S. Typhimurium flagellins in activating TLR5-mediated IL-8 secretion [14,15].
Another potential function of the H. pylori flagellar sheath is the generation of outer membrane vesicles (OMVs). OMVs are released as part of normal bacterial growth and function in a variety of processes, including the delivery of lipopolysaccharide (LPS), proteins, and toxins to target cells; the secretion of enzymes involved in nutrient acquisition; horizontal gene transfer; functioning as decoys for bacteriophages and antibiotics; and the stress response [16,17]. In various Vibrio species, the flagellar sheath has been demonstrated to be a major source of OMVs [18,19]. OMVs derived from the flagellar sheath in Vibrio species have been proposed to originate from membrane blebs along the sheath, which are released as the flagella rotate [18].
For a given bacterial species, knowledge of the proteins that localize to the flagellar sheath is important in understanding the function of the sheath. To this end, several early studies sought to identify the proteins that are present in the sheath [9,11,20,21,22,23,24]. One early study reported that the flagellar sheath of Bdellovibrio bacteriovorus has significantly less protein (23–28% dry weight) than the typical bacterial outer membrane, which generally contains 40–70% protein by dry weight [24]. The reported protein content of the B. bacteriovorus sheath suggests that proteins do not exchange freely between the sheath and outer membrane, which implies that mechanisms are in place that control the protein content of the sheath. Consistent with this hypothesis, Doig and Trust identified six H. pylori protein antigens in the outer membrane that were not detected in the flagellar sheath [20]. Moreover, some proteins have been reported to localize preferentially to the H. pylori flagellar sheath. One such protein is H. pylori adhesion A (HpaA), although there are conflicting reports on the localization of this protein. HpaA was initially observed on the cell surface by immunogold labeling but was not observed on the sheath [25]. A subsequent immunogold labeling study reported that HpaA localized specifically to the sheath [11,26], while another study reported that HpaA was present on both the sheath and cell surface [27]. Another protein that localizes to the H. pylori flagellar sheath is flagellar-associated autotransporter A (FaaA), which was shown by both immunogold labeling and fluorescence microscopy to localize to the sheath [28]. Although the function of FaaA is not known, disrupting faaA resulted in various flagellum-related defects, including decreased motility, reduced flagellation, an increased frequency of broken flagella, and a higher proportion of flagella that localized to nonpolar sites [28].
In an effort to identify proteins with potential roles in the function or biogenesis of the H. pylori flagellar sheath, Gibson and co-workers identified homologs of 42 H. pylori proteins that were widespread in Helicobacter species that possess flagellar sheaths (FS+ species) but were underrepresented in Helicobacter species with sheath-less flagella (FS− species) [29]. One of the proteins that was found preferentially in FS+ Helicobacter species was cardiolipin synthase C (ClsC), which catalyzes the final step in the synthesis of the phospholipid cardiolipin [30]. Cardiolipin consists of four acyl chains attached to a glycerol backbone, which results in the cardiolipin molecule being cone-shaped with a small polar head and a large hydrophobic tail. Cardiolipin molecules form clusters or microdomains that have intrinsic curvature and thus lower energy when localized to regions of the membrane that have negative curvature [31,32]. Consequently, cardiolipin tends to accumulate in membrane regions with negative curvature, such as the cell pole and septum in rod-shaped bacteria [33,34,35,36]. Given that the flagellar sheath is a long, tube-like structure, as well as the proclivity of cardiolipin to accumulate in membranes with negative curvature, one would expect the sheath to contain significant amounts of cardiolipin. Consistent with this hypothesis, the H. pylori flagellar sheath does indeed have significant amounts of cardiolipin [37]. Additional supporting evidence for this hypothesis is the observation that the most abundant fatty acyl chains in the H. pylori flagellar sheath are myristic acid (C14:0) and cyclo-nonadecanoic acid (cycC19:0) [9], which are also the most common fatty acyl chains in H. pylori cardiolipin species [30,38,39].
The disruption of clsC in H. pylori strains G27 and B128 resulted in decreased amounts of cardiolipin, although some cardiolipin was still synthesized in the clsC mutants, indicating that H. pylori strains have at least one more cardiolipin synthase [30]. Escherichia coli has three different cardiolipin synthases, ClsA, ClsB, and ClsC [40,41,42]. Interestingly, the vast majority of the cardiolipin in the H. pylori clsC mutants was in the form of monolysocardiolipin [30], which has three instead of four acyl chains.
We report here that homologs of H. pylori ClsC are restricted to Helicobacter species that have sheathed flagella, and these ClsC homologs are predicted lipoproteins. We further show that a ClsC super-folder green fluorescent protein (ClsC-sfGFP) fusion localized to the cell pole and cell septum in H. pylori G27. Finally, we investigated whether cardiolipin might be required for the localization of some proteins to the H. pylori flagellar sheath, since cardiolipin is required for the polar localization of some proteins in E. coli, including ProP, MscS, and ClsA [36,43]. Comparing the proteomes of flagella preparations from a H. pylori B128 clsC::cat mutant and the wild-type H. pylori B128 parental strain, we identified five proteins in the wild-type flagellum preparations that were not found in the flagella isolated from the clsC::cat mutant. All five proteins are predicted to have signal peptides recognized by the Sec translocon and cleaved by signal peptidase I (Sec/SPI signal peptide), indicating that the proteins are likely secreted across the inner membrane. One of the proteins, FaaA, is an autotransporter that is reported to localize to the flagellar sheath [28]. Taken together, these data suggest that the localization of FaaA and possibly other proteins to the H. pylori flagellar sheath is dependent on ClsC.

2. Materials and Methods

2.1. Bacterial Strains and Growth Conditions

E. coli Turbo (New England Biolabs, Ipswitch, MA, USA) was used for cloning procedures and was cultured in lysogeny broth (LB) and LB agar. Growth media for E. coli strains were supplemented with ampicillin (100 µg/mL) or kanamycin (30 µg/mL) when appropriate. H. pylori G27 (kindly provided by D. Scott Merrell), H. pylori B128 (kindly provided by Richard M. Peck, Jr.), and clsC::cat mutants derived from G27 and B128 [30] were grown on tryptic soy agar supplemented with 5% heat-inactivated horse serum (TSA-HS) and 30 μL/mL kanamycin when appropriate. H. pylori strains on agar medium were grown at 37 °C under atmospheric conditions consisting of 10% CO2, 8% O2, and 82% N2. For liquid cultures, H. pylori strains were grown at 37 °C with shaking in Mueller–Hinton broth (MHB) supplemented with 5% heat-inactivated horse serum under an atmosphere consisting of 5% CO2, 10% H2, 10% O2, and 75% N2.

2.2. Construction of ClsC-sfGFP Fusion

The ClsC-sfGFP was expressed from plasmid pDE43, which is a derivative of the shuttle vector pHel3 [44] and was constructed as follows. Plasmid pHel3-Myc [45] is a derivative of pHel3 that carries the H. pylori fliF promoter upstream of tandem BspQ1 sites used for Golden Gate cloning, which is followed by a sequence encoding a flexible linker, c-Myc epitope, and DDDDK epitope. Plasmid pHel3-GFP was generated by replacing the sequences encoding the c-Myc and DDDDK epitopes in pHel3-Myc with a sequence encoding a sfGFP [46] that was optimized for H. pylori codon usage and synthesized by Azenta Life Sciences (South Plainfield, NJ, USA). Primers sgfp1 (5′-TCGATATCTAGATCTCGAGTTTATTTATACAATTCATCCATGCC-3′) and sgfp2 (5′-TCAGGTGAATTTGCGGCCGCATGAGCAAAGGCGAAGAATTG-3′) were used to amplify the codon-optimized sfGFP gene sequence, and primers phel3-myc1 (5′-ACTCGAGATCTAGATATCGATG-3′) and phel3-myc2 (5′-GCGGCCGCAAATTCACCT-3′) were used to amplify most of plasmid pHel3-Myc. PrimeSTAR Max polymerase (Takara Bio USA Inc, San Jose, CA, USA) was used to amplify the pHel3-Myc and sfGFP DNA sequences as described by the supplier, with the exception of using annealing temperatures of 63 °C and 57 °C, respectively. Following the PCR, the samples were treated with the restriction enzyme DpnI (Promega, Madison, WI, USA) to digest the template DNA, and the amplicons were then transformed into E. coli Turbo cells for in vivo assembly as described in [47] to generate plasmid pHel3-GFP. Primers clsC-BspQ1F (5′-TGGCTCTTCTATGAAAATCTTTTTAGTCCTTTTAAGCGTC-3′) and clsC-BspQ1R (5′-ATGCTCTTCTACCAAGCTCTCTTTCAGGAAGGACT-3′) were used to amplify clsC from H. pylori B128 genomic DNA, and we introduced BspQ1 sites at the ends of the resulting amplicon. PrimeSTAR Max polymerase was used to amplify clsC, following the supplier’s protocol, using an annealing temperature of 64 °C. The clsC amplicon and plasmid pHel3-GFP were digested with pBspQ1 and ligated as described previously [45] to create plasmid pClsC-GFP, which was introduced by natural transformation into H. pylori G27, and the H. pylori G27 clsC::cat mutant and transformants were selected on TSA-HS supplemented with kanamycin.

2.3. Fluorescence Microscopy

One mL overnight cultures of H. pylori strains bearing the plasmid pClsC-GFP were centrifuged at 8700× g for 1 min to pellet the cells. The resulting supernatant liquids were discarded, and the cells were then resuspended in 300 µL of phosphate-buffered saline (PBS). For cells that were stained with the FM4-64 dye, 1 µL of a 10 mM FM4-64 dye stock solution was added to the 1 mL samples of the overnight cultures, and the samples were incubated for 30 min prior to the centrifugation step. Glass microscopy slides were prepared by applying 5 µL of a 10% (w/v) poly-lysine solution to each slide and allowing the slides to air-dry. Seven µL of the resuspended cells was applied to the slide, which was then covered with a 1.5 mm coverslip. Cells were visualized by fluorescence microscopy using a Nikon Ti-U fluorescence microscope equipped with a Lumenocor SOLA SM II light engine. The microscope was fitted with a 100× oil immersion objective (NA 1.45) and the GFP HISM Zero Shift and Texas Red Longpass filter sets. Images were captured using a CooolSNAP My camera (Teledyne Photometrics, Tuscon, AZ, USA) and controlled via the Nikon NIS-Elements BR software package (v. 4.20.1). The images were taken on DIC channel 1 with an exposure time of ~20 ms, GFP channel 3 with an exposure time of ~2 s, and FM464 channel 4 with an exposure time of ~200 ms. The resultant images were processed and analyzed using the ImageJ Fuji software package version 2.14.0/1.5f.

2.4. Mass Spectrometry Analysis of Isolated H. pylori Flagella

H. pylori flagella were isolated and analyzed as described [48]. Briefly, wild-type H. pylori B128 and the H. pylori B128 clsC::cat mutant grown on TSA-HS were resuspended in 3 to 4 mL phosphate-buffered saline (PBS). The cell suspensions were vortexed for 60 s to shear the flagella from the cells, and the cells were subsequently pelleted by centrifugation at 7300× g for 10 min. The resulting supernatants were further clarified by passage through 0.4 µm filters, and the filtrates were centrifuged at 106,000× g for 30 min to pellet the flagella. The flagellar pellets were then resuspended in 200 to 300 μL PBS, and the amounts of protein in the samples were quantified using the Pierce BCA Protein Assay (ThermoFisher Scientific, Waltham, MA, USA), as per the supplier’s instructions. Portions of H. pylori flagella preparations (5 μg total protein) were loaded onto an SDS–polyacrylamide gel and electrophoresed into the top of the resolving gel. Gel slices containing the proteins were sent to the University of Georgia Proteomics and Mass Spectrometry facility for proteomic analysis. Proteins in the gel slices were digested with trypsin by staff at the mass spectrometry facility, and the resulting peptides were analyzed by LC-MS/MS with a ThermoScientific Orbitrap Velo Elite mass spectrometer coupled with nano-HPLC using a 90 min elution gradient. Mass spectrometry data were searched against a protein database using the Mascot software search engine (Matrix Science, Boston, MA, USA) for protein identification.

3. Results

3.1. Homologs of H. pylori ClsC Are Predicted Lipoproteins and Are Restricted to Helicobacter Species That Have Flagellar Sheaths

As indicated above, a survey of 35 FS+ Helicobacter species and 9 FS− Helicobacter species revealed previously that ClsC homologs are prevalent in FS+ species but are absent in FS− species [29]. Given that genome sequences for more Helicobacter species have become available since this earlier survey, we used blastp to search the NCBI non-redundant protein database (accessed on 20 February 2025) and JGI Integrated Microbial Genomes and Microbiomes (IMG/M) database (accessed on 20 February 2025) for homologs of H. pylori G27 ClsC in an additional nine FS+ Helicobacter species and four FS− Helicobacter species. A list of the Helicobacter species examined is indicated in Table S1 along with references for the Helicobacter species [49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96]. For the blastp analysis, an E-value of 1 × 10−20 was used as the cutoff. Of the 44 Helicobacter FS+ species surveyed, 35 had a predicted ClsC homolog (Table S2). All of the ClsC homologs identified in the survey had E-values for the blastp analysis that were <1 × 10−121, which was well below the cutoff value (Table S2). In contrast, ClsC homologs were not identified in any of the 13 FS− Helicobacter species (Table S2). A Fisher’s exact test indicated that the difference in the distribution of ClsC homologs between the FS+ and FS− Helicobacter species was statistically significant (p-value < 0.00001).
The SignalP 6.0 server (https://services.healthtech.dtu.dk/services/SignalP-6.0/; accessed on 14 October 2024) predicts signal peptides in protein sequences from all domains of life. SignalP 6.0 predicted, with a moderately strong likelihood (0.684), that H. pylori B128 ClsC possessed a lipoprotein signal peptide (Table S2). SignalP 6.0 similarly predicted, with a strong likelihood (>0.98), lipoprotein signal peptides for most of the ClsC homologs from the other Helicobacter species identified in our survey (Table S2; accessed on 27 February 2025). A few of the ClsC homologs were predicted to lack a lipoprotein signal peptide, but using an alternative start codon for these proteins identified predicted lipoprotein signal peptides with high likelihood scores (>0.93; Table S2). Importantly, the predicted lipoprotein signal peptides for all of the ClsC homologs had the invariant cysteine residue that is at the cleavage site and is acylated (Figure 1). Taken together, these findings suggest that H. pylori ClsC and the ClsC homologs in other Helicobacter species are lipoproteins.

3.2. H. pylori and Other Helicobacter Species Have a Potential Eukaryotic-Type Cardiolipin Synthase

In addition to ClsC, H. pylori has at least one more cardiolipin synthase, as H. pylori clsC mutants are still able to synthesize cardiolipin [30]. Cardiolipin synthases catalyze the final step in the cardiolipin biosynthetic pathway, and there are two general types of cardiolipin synthases. Bacterial-type cardiolipin synthases have two phospholipase D domains and synthesize cardiolipin by transferring a phosphatidic acid from either phosphatidylglycerol or phosphatidylethanolamine to a phosphatidylglycerol molecule [42,98,99]. Eukaryotic-type cardiolipin synthases possess a cytidine diphosphate (CDP)-alcohol phosphatidyltransferase domain that generates cardiolipin by transferring phosphatidic acid from CDP-diacylglycerol to phosphatidylglycerol [100,101]. H. pylori ClsC is typical of bacterial-type cardiolipin synthases in that it possesses two phospholipase D domains. A search of the H. pylori 26695 genome for candidates for other cardiolipin synthases identified two additional phospholipase D (PLD) family members, HP0323 (NucT) and HP1499. Both NucT and HP1499 have a single phospholipase D domain and belong to the endonuclease PLD subfamily, suggesting that these proteins do not have cardiolipin synthase activity. Moreover, H. pylori NucT is a nuclease associated with the outer membrane and is proposed to be involved in both purine recycling and competence [102,103], while the function of HP1499 is unknown.
Interestingly, a search of the H. pylori 26695 genome identified HP1016 as a CDP-alcohol phosphatidyltransferase domain protein, indicating that this protein may be a cardiolipin synthase. A blastp search of the NCBI non-redundant protein database (accessed on 27 February 2025) and JGI IMG/M database (accessed on 27 February 2025) for homologs of HP1016 in the Helicobacter species that had been surveyed for predicted ClsC homologs identified HP1016 homologs in all but three of the fifty-seven Helicobacter species surveyed (Table S2). In contrast to ClsC, the HP1016 homologs were equally represented in FS+ and FS− Helicobacter species. The HP1016 homologs from the various Helicobacter species were predicted by the Phobius server (https://phobius.sbc.su.se/; accessed on 15 April 2025) to have three to six transmembrane (TM) helices (Table S2), indicating that they are likely integral inner membrane proteins.
To determine if HP1016 is indeed a cardiolipin synthase, we attempted to delete the hp1016 homolog in both a H. pylori B128 clsC::cat mutant and its wild-type parental strain with the intention of examining the phospholipid content of the resulting mutants. Despite several attempts, we were unable to knock out the hp1016 homolog in either of the two H. pylori B128 strains and were therefore unable to determine if HP1016 has cardiolipin synthase activity using our planned approach. The failure to disrupt the hp1016 homolog in H. pylori B128 suggests that the gene is essential.

3.3. ClsC Localizes to the Cell Pole and Cell Septum in H. pylori

As a predicted lipoprotein, H. pylori ClsC could localize to either the inner membrane or outer membrane. Zavan and co-workers reported that ClsC was one of the top thirty proteins that were significantly more abundant in H. pylori OMVs compared to the global proteome [104], which strongly suggests that ClsC localizes to the outer membrane. As indicated previously, ClsA co-localizes with cardiolipin to the cell pole in E. coli [43]. To determine if H. pylori ClsC similarly localizes to specific regions of the cell, we examined the localization of a ClsC-sfGFP fusion in H. pylori G27 using fluorescence microscopy. For the initial analysis, we examined the localization of the ClsC-sfGFP fusion in a clsC::cat mutant to avoid possible competition between the ClsC-sfGFP fusion and native ClsC for binding sites within the cell. Fluorescent foci attributed to the ClsC-sfGFP fusion were observed near the cell poles in virtually all cells, as well as near the midcell in many of the cells (Figure 2). Scanning fluorescence microscopy images of several of the cells confirmed the presence of distinct fluorescent foci within the cells (Figure 2D–I).
The localization of the ClsC-sfGFP fusion near the midcell in many of the cells suggested that the fusion protein was recruited to the cell septum. To examine the validity of this hypothesis, we compared the lengths of cells that had fluorescent foci only at the cell poles versus cells that had fluorescent foci at both the cell poles and the midcell. The average length of cells that had midcell fluorescent foci was 2.72 ± 0.15 µm (n = 50), while average length of cells that had fluorescent foci only at the cell poles was 1.52 ± 0.034 µm (n = 50). The difference in cell length between the two cell types was significant, as determined using a Mann–Whitney U test (p = 0.00008). We inferred from these data that the longer cells were undergoing cell division and the ClsC-sfGFP fusion localized to the cell septum in these cells. To address further the hypothesis that the ClsC-sfGFP fusion localizes to the cell septum, we stained H. pylori cells with the lipophilic fluorescent dye FM4-64 to visualize the membranes. The staining of H. pylori cells with FM4-64 revealed that fluorescent foci due to the ClsC-sfGFP fusion that localized near the midcell were closely associated with membranes that, in some cases, appeared to bisect the cell (Figure 3C,D). In cells that had two distinct fluorescent foci at the midcell, the foci appeared to be separated by the membrane that bisected the cell. We postulate that the membrane separating the two fluorescent foci is part of the cell septum.
The examination of wild-type H. pylori G27 cells expressing the ClsC-sfGFP fusion revealed that they similarly displayed distinct fluorescent foci near the cell poles and midcell (Figure 3F,H), indicating that the presence of native ClsC does not interfere with the localization of the ClsC-sfGFP fusion to these sites. As observed with the clsC::cat mutant, fluorescent foci near the midcell in the wild-type cells were associated with membranes that appeared to bisect the cell (Figure 3H).

3.4. The Presence of Some Proteins Within the H. pylori Flagellar Sheath Appears to Be Dependent on ClsC

Given that ProP, MscS, and ClsA co-localize with cardiolipin to the cell pole in E. coli [36,43], we wished to determine if the localization of some proteins to the H. pylori flagellar sheath was dependent on cardiolipin. For this study, we used a H. pylori B128 clsC::cat mutant, since the H. pylori G27 clsC mutant is aflagellated, while the H. pylori B128 clsC mutant displays wild-type flagellation and motility in soft agar medium [30]. The molecular basis for the difference in flagellation between the H. pylori G27 clsC mutant and H. pylori B128 clsC mutant is not known, but replacing the flgI (which encodes the flagellar P-ring protein) allele in the H. pylori G27 clsC mutant with the H. pylori B128 flgI allele rescues flagellum biogenesis [30]. Although the H. pylori B128 clsC::cat mutant still synthesizes small amounts of cardiolipin, most of the cardiolipin in the mutant is in the form of monolysocardiolipin [30], and we reasoned that the depletion of cardiolipin in the clsC::cat mutant may be sufficient to alter the partitioning of some proteins to the sheath. To identify flagellar sheath proteins that may be dependent on cardiolipin for their localization, we compared the proteomes of flagella prepared from wild-type H. pylori B128 and the H. pylori B128 clsC::cat mutant. Flagella that were sheared from the H. pylori cells were isolated with their accompanying sheaths and analyzed by mass spectroscopy in two biological replicates for both the wild type and the clsC mutant.
A combined total of 190 non-ribosomal proteins were identified in at least two of the samples (Table S3). The proteins listed in Table S3 are ordered by their protein scores for the first biological replicate of the clsC::cat mutant. The Mascot software package from Matrix Science (www.matrixscience.com; accessed on 13 September 2023) was used to identify the proteins from the mass spectral data. Mascot calculates a protein score from the combined scores of all observed mass spectra, and the score indicates the confidence for the protein identification. Although the protein scores are not quantitative for protein levels, the scores often correlate with the relative abundance of proteins. In all four samples, the major flagellin (FlaA), minor flagellin (FlaB), hook protein (FlgE), and hook-associated proteins (FlgL and FlgK) had some of the highest protein scores (Table S3). Sixty-four percent (121 of 190) of the identified proteins were known or predicted to be associated with the flagellum, flagellar sheath, outer membrane, or periplasmic space (Table S3). The remaining proteins were either predicted inner membrane or cytoplasmic proteins. With the exception of a few highly expressed proteins, such as the urease and GroEL/GroES chaperone, the Mascot protein scores were relatively low for most of the cytoplasmic proteins. Thus, the proportion of proteins known or predicted to be associated with the flagellum, flagellar sheath, outer membrane, or periplasmic space increased to 84% when considering the 100 proteins with the highest protein scores. Taken together, these data indicate that the flagellar preparations were highly enriched for flagellar, sheath, outer membrane, and periplasmic proteins.
Five proteins were present in both replicates of the wild-type flagellar preparation but were not detected in the flagellar preparations from the clsC::cat mutant (Table 1). Two of the proteins, HofH and FaaA, are known outer membrane proteins and are therefore promising candidates for proteins that are dependent on ClsC for their localization to the flagellar sheath. HofH is an outer membrane β-barrel protein, while FaaA is an autotransporter that shares homology with the pore-forming VacA toxin and localizes to the flagellar sheath [28].
The other three proteins present in both replicates of the wild-type flagellar preparation but not detected in the isolated flagella of the clsC::cat mutant (HP0519, HP0408, and HP0709) were predicted by SignalP 6.0 to have Sec/SPI signal peptides—the likelihood scores for HP0519, HP0408, and HP0709 were 0.9988, 0.9989, and 0.9279, respectively (accessed on 1 April 2025). Thus, it is very likely that these proteins are secreted across the inner membrane. The function of HP0519 is unknown, but it belongs to a family of Helicobacter proteins that contain two or more copies of a degenerate 34–36-amino-acid repeat motif that is characteristic of eukaryotic Sel1 proteins [105]. The function of HP0408 is also unknown. The AlphaFold database feature in the Dali server (http://ekhidna2.biocenter.helsinki.fi/dali/ accessed on 15 April 2025) indicated that the predicted tertiary structure of HP0408 shared significant homology with E. coli ModA (Z-score = 10.6 accessed on 15 April 2025). ModA is a periplasmic molybdate-binding protein, and the structural homology of the proteins indicates that HP0408 may be a periplasmic binding protein. The function of HP0709 is also unknown, but it belongs to a family of proteins that includes enzymes that catalyze nucleophilic reactions of fluoride or chloride ions to the C-5′ carbon of S-adenosyl-L-methionine (SAM) [106]. If HP0709 is a periplasmic protein, it is doubtful that it is a SAM-utilizing enzyme, since SAM would not be present in the periplasm.

4. Discussion

While the sheathed flagellum of H. pylori is a distinctive feature of this bacterial pathogen, the function and biogenesis of the bacterium’s flagellar sheath remains enigmatic. The biogenesis of the flagellar sheath requires the mobilization of both lipids and proteins to the flagellated cell pole for their incorporation into the nascent sheath. One phospholipid required for sheath biogenesis is cardiolipin, which accumulates in membrane regions with negative curvature, such as the sheath, due to the capacity of cardiolipin molecules to form microdomains that have intrinsic curvature [31,32]. H. pylori ClsC and homologs of the enzyme present in other Helicobacter species are predicted to be lipoproteins (Table S2), and this feature is likely important in determining the subcellular localization of ClsC within the bacterial cell.
Although we did not detect ClsC in the preparations of isolated flagella from wild-type H. pylori B128 (Table S3), ClsC was reported to be highly enriched in H. pylori OMVs compared to the global proteome [104], which strongly suggests that ClsC localizes to the outer membrane. Our failure to detect ClsC in the proteomes of the isolated flagella may be due to the enzyme being in low abundance. Flagellar sheath biogenesis exhibits a significant demand for cardiolipin in the outer membrane, and a potential advantage of the localization of ClsC to the outer membrane is that it circumvents the need for the anterograde transport of cardiolipin (i.e., transport from the inner membrane to the outer membrane). The observation that ClsC homologs appear to be restricted to Helicobacter species that have sheathed flagella (Table S2) supports the notion that synthesizing cardiolipin within the outer membrane imparts a physiological advantage in sheath biogenesis. The biosynthesis of cardiolipin within the outer membrane would allow cardiolipin to be rapidly incorporated into the nascent flagellar sheath. More importantly, however, synthesizing cardiolipin in the outer membrane would avoid competition between cardiolipin and other phospholipids in anterograde transport. AsmA-like proteins are proposed to be responsible for the majority of phospholipid anterograde transport [107]. AsmA-like proteins have an N-terminal α-helix that anchors the protein in the inner membrane and a large periplasmic domain composed of repeating β-taco domains that form a long β-groove with a hydrophobic interior [107]. The hydrophobic groove of AsmA-like proteins likely shields the fatty acids of phospholipids as they diffuse across the periplasm. E. coli possesses six AsmA-like proteins (AsmA, TamB, YhdP, YdbH, YhjG, and YicH) [107], while H. pylori has only two AsmA-like proteins (HP0586 and HP0358). It would be interesting to determine if replacing ClsC with a cardiolipin synthase that resides in the inner membrane [e.g., E. coli ClsA] impacts the kinetics of flagellar sheath biogenesis or perhaps affects cell fitness by placing an added burden on the anterograde transport of phospholipids.
The ClsC-sfGFP fusion localized to the cell pole and cell septum (Figure 2 and Figure 3), which may have been consequential, since the membrane regions in these areas of the cell are where were cardiolipin accumulates. Cardiolipin is required for the polar localization of some proteins in E. coli [36,43], and it is possible that the localization of ClsC to the cell pole and septum is similarly dependent on cardiolipin. Alternatively, ClsC may recognize a landmark protein or engage another protein that localizes to the cell pole and septum. Future experiments designed to identify ClsS interaction partners may shed light on the molecular basis for the localization of ClsC to the cell pole and septum, as well as identifying proteins that may regulate the activity of ClsC.
Finally, a significant gap in our knowledge of sheath biogenesis and function is understanding how outer membrane proteins are partitioned between the sheath and outer membrane. Comparing the proteomes of isolated flagella from the wild type and the clsC mutant, we identified five proteins (FaaA, HofH, HP0519, HP0408, and HP0709) whose presence in the flagella preparations appeared to be dependent on ClsC (Table 1 and Table S3). It seems unlikely that the failure to detect these proteins in the isolated flagella of the clsC mutant was due to the reduced expression of the proteins, as a previous transcriptome analysis indicated the transcript levels of the genes encoding the proteins were comparable in the wild type and the H. pylori G27 clsC mutant [30]. The failure to identify FaaA in the isolated flagella of the clsC mutant is of particular interest since FaaA localizes predominantly in the flagellar sheath [28]. The Sel1 family protein HP0519 is also of interest since it appeared to be relatively abundant in the wild-type flagellar samples (based on the Mascot score, number of unique peptides, and percent coverage for the protein) but was undetected in the isolated flagella of the clsC::cat mutant (Table 1 and Table S3). We are unaware of any reports on the subcellular localization of HP0519, but we are working currently to address this issue. We are also working to verify that the localization of FaaA is dependent on ClsC by using fluorescently labeled antibodies to the protein and examining the H. pylori cells by fluorescence microscopy. While it is possible that ClsC plays a direct role in affecting the localization of FaaA and HofH to the sheath, it seems more likely that the failure of these proteins to localize to the sheath is due to the depletion of cardiolipin in the sheath. If the localization of FaaA and/or HofH to the sheath is dependent on cardiolipin, it is likely that these proteins have an affinity for cardiolipin microdomains within the sheath.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microbiolres16070155/s1, Table S1: Helicobacter species examined for homologs of ClsC and HP1016; Table S2: Homologs of ClsC and HP1016 in Helicobacter species; Table S3: Proteins identified in the flagellar preparations of wild-type H. pylori B128 and H. pylori B128 clsC mutant.

Author Contributions

Conceptualization, T.R.H. and D.N.; investigation, D.N. and N.E.; writing—original draft preparation, D.N.; writing—review and editing, T.R.H. and V.J.S.; visualization, D.N. and N.E.; supervision, T.R.H. and V.J.S.; project administration, T.R.H.; funding acquisition, T.R.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Institutes of Health, grant number AI140444. The proteomics data generated in the study were supported by instrument grant NIH S10 OD025118 to the UGA PAMS facility.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article and Supplementary Materials; further inquiries can be directed to the corresponding author.

Acknowledgments

We thank Robert Maier and Stéphane Benoit for the use of the equipment and laboratory space and thank Lindsay Berardi for the technical assistance.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
sfGFPSuper-folder green fluorescent protein
TLR5Toll-like receptor 5
IL-8Interleukin-8
OMVOuter membrane vesicle
LPSLipopolysaccharide
FS+Flagellar sheath positive
FS−Flagellar sheath negative
SPISignal peptidase I
PLDPhospholipase D
TMTransmembrane
SAMS-adenosyl-L-methionine
TSA-HSTryptic soy agar supplemented with horse serum
MHBMueller–Hinton broth
MES2-(N-morpholino)ethanesulfonic acid
PBSPhosphate-buffered saline
SDSSodium dodecyl sulfate
CDPCytidine diphosphate
LCLiquid chromatography
HPLCHigh-performance liquid chromatography
MSMass spectrometry
NCBINational Center for Biotechnology Information
JGIJoint Genome Institute
IMG/MIntegrated Microbial Genomes and Microbiomes

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Figure 1. N-terminal sequences of H. pylori ClsC and select ClsC homologs from other Helicobacter species. The backslash indicates the predicted cleavage site at the invariant cysteine residue of the lipoprotein signal peptide. The invariant cysteine together with the three proceeding amino acid residues constitute a motif referred to as the lipobox (indicated in red), which is important for the recognition and processing of the apolipoprotein. The consensus sequence for the lipobox is [LVI][ASTVI][GAS][C] [97]. For the Helicobacter bilis ClsC homolog, using the methionine that is underlined and in boldface as the N-terminal residue of the protein changed the likelihood score for a lipoprotein signal peptide from 0.0026 to 1.
Figure 1. N-terminal sequences of H. pylori ClsC and select ClsC homologs from other Helicobacter species. The backslash indicates the predicted cleavage site at the invariant cysteine residue of the lipoprotein signal peptide. The invariant cysteine together with the three proceeding amino acid residues constitute a motif referred to as the lipobox (indicated in red), which is important for the recognition and processing of the apolipoprotein. The consensus sequence for the lipobox is [LVI][ASTVI][GAS][C] [97]. For the Helicobacter bilis ClsC homolog, using the methionine that is underlined and in boldface as the N-terminal residue of the protein changed the likelihood score for a lipoprotein signal peptide from 0.0026 to 1.
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Figure 2. Differential interference contrast (DIC) and fluorescence microscopy images of H. pylori cells expressing a ClsC-sfGFP fusion protein. (A) DIC microscopy image of a field of cells in the H. pylori G27 clsC::cat mutant expressing the ClsC-sfGFP fusion. (B) Fluorescence microscopy image of the field of H. pylori cells shown in panel (A). (C) Merger of the DIC microscopy and fluorescence microscopy images shown in panels (A,B), respectively. (D–F) Fluorescence microscopy images of individual H. pylori cells expressing the ClsC-sfGFP fusion protein. Fluorescence microscopy images of H. pylori cells displaying four (D), three (E), or two (F) fluorescent foci were scanned and analyzed using the ImageJ Fuji software package version 2.14.0/1.5f. (G–I) Graphic displays from each scan are presented below the corresponding fluorescence microscopy image. In the graphs shown here, the scans extend beyond the lengths of the cells.
Figure 2. Differential interference contrast (DIC) and fluorescence microscopy images of H. pylori cells expressing a ClsC-sfGFP fusion protein. (A) DIC microscopy image of a field of cells in the H. pylori G27 clsC::cat mutant expressing the ClsC-sfGFP fusion. (B) Fluorescence microscopy image of the field of H. pylori cells shown in panel (A). (C) Merger of the DIC microscopy and fluorescence microscopy images shown in panels (A,B), respectively. (D–F) Fluorescence microscopy images of individual H. pylori cells expressing the ClsC-sfGFP fusion protein. Fluorescence microscopy images of H. pylori cells displaying four (D), three (E), or two (F) fluorescent foci were scanned and analyzed using the ImageJ Fuji software package version 2.14.0/1.5f. (G–I) Graphic displays from each scan are presented below the corresponding fluorescence microscopy image. In the graphs shown here, the scans extend beyond the lengths of the cells.
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Figure 3. Fluorescence microscopy of H. pylori cells stained with FM4-64 dye. (A) DIC microscopy image of a H. pylori G27 clsC::cat mutant cell stained with FM4-64 dye. (B) Fluorescence microscopy of H. pylori cell shown in panel (A) viewed in the green (GFP) channel. Fluorescent foci are present near each cell pole, and two fluorescent foci are present near the midcell. (C) Fluorescence microscopy image of H. pylori cell shown in panel (A) viewed in the red (FM4-64) channel. (D) Merger of the fluorescence microscopy images shown in panels (B,C). (E) DIC microscopy image of wild-type H. pylori G27 cells expressing the ClsC-sfGFP fusion that were stained with FM4-64 dye. (F) Fluorescence microscopy image of H. pylori cells shown in panel (E) viewed in the green channel. (G) Fluorescence microscopy image of H. pylori cells shown in panel (E) viewed in the red channel. (H) Merger of the fluorescence microscopy images shown in panels (F,G). The arrows in panels (C,D,G,H) indicate putative cell septa.
Figure 3. Fluorescence microscopy of H. pylori cells stained with FM4-64 dye. (A) DIC microscopy image of a H. pylori G27 clsC::cat mutant cell stained with FM4-64 dye. (B) Fluorescence microscopy of H. pylori cell shown in panel (A) viewed in the green (GFP) channel. Fluorescent foci are present near each cell pole, and two fluorescent foci are present near the midcell. (C) Fluorescence microscopy image of H. pylori cell shown in panel (A) viewed in the red (FM4-64) channel. (D) Merger of the fluorescence microscopy images shown in panels (B,C). (E) DIC microscopy image of wild-type H. pylori G27 cells expressing the ClsC-sfGFP fusion that were stained with FM4-64 dye. (F) Fluorescence microscopy image of H. pylori cells shown in panel (E) viewed in the green channel. (G) Fluorescence microscopy image of H. pylori cells shown in panel (E) viewed in the red channel. (H) Merger of the fluorescence microscopy images shown in panels (F,G). The arrows in panels (C,D,G,H) indicate putative cell septa.
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Table 1. Proteins identified in the wild-type H. pylori B128 flagellar preparations but not the flagellar preparations from the H. pylori B128 clsC::cat mutant.
Table 1. Proteins identified in the wild-type H. pylori B128 flagellar preparations but not the flagellar preparations from the H. pylori B128 clsC::cat mutant.
First Biological ReplicateSecond Biological Replicate
Locus TagDescriptionScore# Peptides% CoverageScore# Peptides% Coverage
HP0519sel1 repeat family protein929.181144.52911.841140.99
HP1167OM protein HofH912.411330.36317.34513.59
HP0408hypothetical protein95.07329.93174.46549.64
HP0709hypothetical protein175.28413.67258.20414.67
HP0609autotransporter FaaA147.0520.66532.3982.61
The “Score” indicates the Mascot score for the protein, “# peptides” indicates the number of unique peptide sequences identified for the protein, and “% coverage” refers to the fraction of the protein’s amino acid sequence that is covered by the identified peptides.
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Nguyen, D.; East, N.; Starai, V.J.; Hoover, T.R. Localization of a Cardiolipin Synthase in Helicobacter pylori and Its Impact on the Flagellar Sheath Proteome. Microbiol. Res. 2025, 16, 155. https://doi.org/10.3390/microbiolres16070155

AMA Style

Nguyen D, East N, Starai VJ, Hoover TR. Localization of a Cardiolipin Synthase in Helicobacter pylori and Its Impact on the Flagellar Sheath Proteome. Microbiology Research. 2025; 16(7):155. https://doi.org/10.3390/microbiolres16070155

Chicago/Turabian Style

Nguyen, Doreen, Nathan East, Vincent J. Starai, and Timothy R. Hoover. 2025. "Localization of a Cardiolipin Synthase in Helicobacter pylori and Its Impact on the Flagellar Sheath Proteome" Microbiology Research 16, no. 7: 155. https://doi.org/10.3390/microbiolres16070155

APA Style

Nguyen, D., East, N., Starai, V. J., & Hoover, T. R. (2025). Localization of a Cardiolipin Synthase in Helicobacter pylori and Its Impact on the Flagellar Sheath Proteome. Microbiology Research, 16(7), 155. https://doi.org/10.3390/microbiolres16070155

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