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Cited by 1Bacterial Genetics and BiotechnologyHelicobacter pylori-related gastroenterology studiesChemokine receptors and signaling

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The Helicobacter pylori TlpD cytoplasmic chemoreceptor requires an intact C-terminus for polar localization and function

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Lopez-Magaña, Raymondo, Ottemann, Karen M.·2026·Journal of Bacteriology
DOI 10.1128/jb.00394-25PMC PMC13001251

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Abstract

Bacteria localize proteins to distinct subcellular locations, including chemoreceptors, which frequently localize to the bacterial pole. Although some polarity-promoting mechanisms have been described, many chemoreceptors lack clear routes to becoming polar. TlpD of the bacterial pathogen Helicobacter pylori is one such protein. This cytoplasmic chemoreceptor localizes to the pole in a manner that is independent of the other chemoreceptors. In this work, we evaluated the role of TlpD domains in its function. Truncated proteins were created that lacked different amounts of the N- or C-termini and expressed in H. pylori in place of native tlpD or as the sole chemoreceptor. These TlpD variants were examined for their presence and abundance, protein localization, association with chemotaxis signaling proteins, and effect on motility. TlpD that lacked any portion of the N-terminal 104 amino acids produced low to no amounts of detectable protein. In contrast, TlpD was detectable with loss of the C-terminal 45 amino acids. TlpD lacking the last 45 amino acids (TlpD∆C4) preserved the ability to interact with CheW and CheV proteins based on bacterial two-hybrid analysis, but was unable to localize to the pole either on their own or in the presence of other chemoreceptors. TlpD∆C4 was found to be diffuse in the cytoplasm and interacted with CheV1, CheV2, and CheV3 at this location but not with CheW. TlpD∆C4 did not confer chemotactic abilities in soft agar chemotaxis assays. These findings suggest the C-terminal end of TlpD plays a previously unappreciated role in promoting TlpD polar localization and function. IMPORTANCE: Bacteria place their proteins in specific locations that are required for the proteins to function, including the bacterial pole. How the bacterial cell identifies which proteins go to the pole is not fully understood. In this work, we dissect parts of a protein called TlpD that naturally goes to the pole. We find that mutants lacking one end of TlpD lose their polar placement, but retain other abilities. TlpD allows directed motility known as chemotaxis. This ability is critical for infection in Helicobacter pylori and numerous other pathogens. When TlpD loses its polar placement, the protein no longer functions for chemotaxis, laying the foundation for future studies that can dissect how this segment promotes function and eventually translate into therapies for H. pylori infection.

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Figures

Fig 1 H. pylori TlpD predicted structure and domains. (A) AlphaFold predicted structure of H. pylori TlpD. TlpD is 433 amino acids in length with two known domains, the MA domain (orange) and the CZB domain (blue). The region with no known domains at the N-terminus is shown in green, and at the C-terminus in red. (B) Diagram of modified versions of H. pylori TlpD with domains and truncation sites indicated (gray). TlpD represents the full-length version of the protein at 433 amino acids, ΔN4 is truncated at residue 53, ΔN5 is truncated at residue 104, ΔC3 is truncated at residue 322, and ΔC4 is truncated at residue 388. Each of these constructs contains a C-terminal 3xFLAG tag.
Fig 1 H. pylori TlpD predicted structure and domains. (A) AlphaFold predicted structure of H. pylori TlpD. TlpD is 433 amino acids in length with two known domains, the MA domain (orange) and the CZB domain (blue). The region with no known domains at the N-terminus is shown in green, and at the C-terminus in red. (B) Diagram of modified versions of H. pylori TlpD with domains and truncation sites indicated (gray). TlpD represents the full-length version of the protein at 433 amino acids, ΔN4 is truncated at residue 53, ΔN5 is truncated at residue 104, ΔC3 is truncated at residue 322, and ΔC4 is truncated at residue 388. Each of these constructs contains a C-terminal 3xFLAG tag.CC BY · PubMed Central
Fig 2 Analysis of abundance of TlpD WT and truncated variants encoded in H. pylori rdxA. (A) Western blot of H. pylori G27 ∆tlpD strains complemented with tlpD constructs was analyzed from whole-cell lysates with an anti-FLAG antibody, which recognizes the C-terminal TlpD tag. The TlpD variant is indicated at the top, with TlpD indicating full-length with the 3× FLAG. Marker sizes are given in kilodaltons on the right. The expected size of TlpD is 51 kDa, ΔN4 is 45 kDa, ΔN5 is 39 kDa, ΔC3 is 38 kDa, and ΔC4 is 45 kDa. Western blot is representative of three biological replicates. (B) Relative protein intensity from western blots. The TlpD variant quantified is indicated below, and the relative density is relative to full-length TlpD. The relative density is calculated from three biological replicates with error bars indicating the standard deviation.
Fig 2 Analysis of abundance of TlpD WT and truncated variants encoded in H. pylori rdxA. (A) Western blot of H. pylori G27 ∆tlpD strains complemented with tlpD constructs was analyzed from whole-cell lysates with an anti-FLAG antibody, which recognizes the C-terminal TlpD tag. The TlpD variant is indicated at the top, with TlpD indicating full-length with the 3× FLAG. Marker sizes are given in kilodaltons on the right. The expected size of TlpD is 51 kDa, ΔN4 is 45 kDa, ΔN5 is 39 kDa, ΔC3 is 38 kDa, and ΔC4 is 45 kDa. Western blot is representative of three biological replicates. (B) Relative protein intensity from western blots. The TlpD variant quantified is indicated below, and the relative density is relative to full-length TlpD. The relative density is calculated from three biological replicates with error bars indicating the standard deviation.CC BY · PubMed Central
Fig 3 TlpD’s interaction with chemotaxis proteins is retained when the C-terminus is truncated. BACTH β-galactosidase (β-gal) assays of full-length and truncated (TlpDΔC4) TlpD with CheV1 and CheW. (A) Diagrams of constructs used. (B) β-gal assays. Positive (+) controls are T25-zip and T18-zip. Negative (−) controls are the respective TlpD construct with blank T25 plasmids. β-gal activity is calculated from three technical replicates with error bars indicating the standard error of the mean. *P < 0.05 and ****P < 0.0001.
Fig 3 TlpD’s interaction with chemotaxis proteins is retained when the C-terminus is truncated. BACTH β-galactosidase (β-gal) assays of full-length and truncated (TlpDΔC4) TlpD with CheV1 and CheW. (A) Diagrams of constructs used. (B) β-gal assays. Positive (+) controls are T25-zip and T18-zip. Negative (−) controls are the respective TlpD construct with blank T25 plasmids. β-gal activity is calculated from three technical replicates with error bars indicating the standard error of the mean. *P < 0.05 and ****P < 0.0001.CC BY · PubMed Central
Fig 4 TlpD is polar when the C-terminus is intact and diffused when the C-terminus is deleted. Immunolocalization of TlpD or TlpD∆C4 in H. pylori whole cells. (A, B) H. pylori was detected with anti-H. pylori (red). TlpD proteins indicated above each image were detected with antibodies that detect all chemoreceptors (anti-TlpA22 or anti-FLAG (green). Scale bar = 4 μM. Images are representative of three biological replicates, with 250–300 bacterial cells analyzed for each sample. (C) Quantification of TlpD immunolocalization from the 250–300 cells are displayed as a percentage of cells with either only polar and no cytoplasmic staining (polar), or both polar and cytoplasmic staining (polar and diffuse).
Fig 4 TlpD is polar when the C-terminus is intact and diffused when the C-terminus is deleted. Immunolocalization of TlpD or TlpD∆C4 in H. pylori whole cells. (A, B) H. pylori was detected with anti-H. pylori (red). TlpD proteins indicated above each image were detected with antibodies that detect all chemoreceptors (anti-TlpA22 or anti-FLAG (green). Scale bar = 4 μM. Images are representative of three biological replicates, with 250–300 bacterial cells analyzed for each sample. (C) Quantification of TlpD immunolocalization from the 250–300 cells are displayed as a percentage of cells with either only polar and no cytoplasmic staining (polar), or both polar and cytoplasmic staining (polar and diffuse).CC BY · PubMed Central
Fig 5 Localization of CheW and CheV coupling proteins in H. pylori in the presence of TlpDΔC4. (A) Immunolocalization of H. pylori chemotaxis proteins CheW, CheV1, CheV2, and CheV3 in whole cells producing full-length (TlpD-FLAG) or truncated (TlpDΔC4-FLAG) protein. H. pylori was detected with chicken anti-H. pylori (red). Individual chemotaxis proteins indicated on the left were detected with preabsorbed guinea pig anti-CheW, rabbit anti-CheV1, rabbit anti-CheV2, or rabbit anti-CheV3 (green). Scale bar = 4 μM. Images are representative of three biological replicates, with 250–300 bacterial cells analyzed for each sample. (B, C) Quantification of Immunolocalization results are displayed as a percentage of polar or polar and diffuse cells as in Fig. 4.
Fig 5 Localization of CheW and CheV coupling proteins in H. pylori in the presence of TlpDΔC4. (A) Immunolocalization of H. pylori chemotaxis proteins CheW, CheV1, CheV2, and CheV3 in whole cells producing full-length (TlpD-FLAG) or truncated (TlpDΔC4-FLAG) protein. H. pylori was detected with chicken anti-H. pylori (red). Individual chemotaxis proteins indicated on the left were detected with preabsorbed guinea pig anti-CheW, rabbit anti-CheV1, rabbit anti-CheV2, or rabbit anti-CheV3 (green). Scale bar = 4 μM. Images are representative of three biological replicates, with 250–300 bacterial cells analyzed for each sample. (B, C) Quantification of Immunolocalization results are displayed as a percentage of polar or polar and diffuse cells as in Fig. 4.CC BY · PubMed Central
Fig 6 H. pylori that produce TlpDΔC4 as the sole chemoreceptor do not spread on soft agar. (A) H. pylori wild-type mG27 and isogenic mutants were analyzed for migration in soft agar. Strains were stabbed onto soft agar, and spreading was measured after 3 days. Strains included are the parent H. pylori lacking all chemoreceptors (∆abcd), and isogenic versions complemented with a full-length TlpD (rdxA::tlpD) (A) or with TlpDΔC4 (rdxA::tlpD ΔC4 3xflag) (B). Plates are representative of three biological replicates, with three technical triplicates. Error bars represent the standard error of the mean.
Fig 6 H. pylori that produce TlpDΔC4 as the sole chemoreceptor do not spread on soft agar. (A) H. pylori wild-type mG27 and isogenic mutants were analyzed for migration in soft agar. Strains were stabbed onto soft agar, and spreading was measured after 3 days. Strains included are the parent H. pylori lacking all chemoreceptors (∆abcd), and isogenic versions complemented with a full-length TlpD (rdxA::tlpD) (A) or with TlpDΔC4 (rdxA::tlpD ΔC4 3xflag) (B). Plates are representative of three biological replicates, with three technical triplicates. Error bars represent the standard error of the mean.CC BY · PubMed Central

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Research works printed but not linked: 1

  1. Castellón J. 2013. CheV1 leads the organization of chemotaxis signal transduction proteins in Helicobacter pylori Masters Thesis University of California Santa Cruz

Metadata

Authors
Lopez-Magaña, R., & Ottemann, K. M.
Affiliations
University of California, Santa Cruz (US)
Corresponding author
Ottemann, Karen M.
ORCID
Venue
Journal of Bacteriology
Publisher
American Society for Microbiology
Published
2026-03-19
Volume
208
Issue
3
Pages
e0039425-e0039425
ISSN
  • 0021-9193 (ISSN-L)
  • 1098-5530
DOI
10.1128/jb.00394-25
PMC
PMC13001251
OpenAlex
W7128395484
Cited by
1
References
63
Citations by year
2026: 1
FWCI
3.96
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Bacteriology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • PubMed Central (submittedVersion)
Funders
National Institutes of Health (RO1AI116946)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Health Sciences › Medicine › Gastroenterology › Helicobacter pylori-related gastroenterology studies
  • Health Sciences › Medicine › Oncology › Chemokine receptors and signaling
Keywords
Helicobacter pylori, polar localization, C-terminal domain, chemotaxis, bacterial motility, CheW, chemoreceptor signaling, infection pathogenesis

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