Crash landing of Vibrio cholerae by MSHA pili-assisted braking and anchoring in a viscoelastic environment
Abstract
Mannose-sensitive hemagglutinin (MSHA) pili and flagellum are critical for the surface attachment of Vibrio cholerae, the first step of V. cholerae colonization on host surfaces. However, the cell landing mechanism remains largely unknown, particularly in viscoelastic environments such as the mucus layers of intestines. Here, combining the cysteine-substitution-based labelling method with single-cell tracking techniques, we quantitatively characterized the landing of V. cholerae by directly observing both pili and flagellum of cells in a viscoelastic non-Newtonian solution consisting of 2% Luria-Bertani and 1% methylcellulose (LB+MC). The results show that MSHA pili are evenly distributed along the cell length and can stick to surfaces at any point along the filament. With such properties, MSHA pili are observed to act as a brake and anchor during cell landing which include three phases: running, lingering, and attaching. Importantly, loss of MSHA pili results in a more dramatic increase in mean path length in LB+MC than in 2% LB only or in 20% Ficoll solutions, indicating that the role of MSHA pili during cell landing is more apparent in viscoelastic non-Newtonian fluids than viscous Newtonian ones. Our work provides a detailed picture of the landing dynamics of V. cholerae under viscoelastic conditions, which can provide insights into ways to better control V. cholerae infections in real mucus-like environment.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1-6.
Article and author information
Author details
Funding
National Key R and D Program of China (2018YFA0902102)
- Kun Zhao
National Natural Science Foundation of China (31770132)
- Zhi Liu
National Natural Science Foundation of China (81572050)
- Zhi Liu
National Natural Science Foundation of China (21621004)
- Kun Zhao
University of Bristol (Vice-Chancellor's Fellowship)
- Rachel R Bennett
Grant in aid for Young Scientists (17K15410)
- Andrew S Utada
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Raymond E Goldstein, University of Cambridge, United Kingdom
Ethics
Animal experimentation: All mice received the humane care and the experimental protocols were carried out in accordance with the Guide for the Care and Use of Laboratory Animals, Huazhong University of Science and Technology, as approved by the Animal Care Committee of Hubei Province.
Version history
- Received: July 2, 2020
- Accepted: July 1, 2021
- Accepted Manuscript published: July 2, 2021 (version 1)
- Version of Record published: July 15, 2021 (version 2)
Copyright
© 2021, Zhang et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,230
- Page views
-
- 177
- Downloads
-
- 5
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Microbiology and Infectious Disease
It is currently unknown whether all Plasmodium falciparum-infected mosquitoes are equally infectious. We assessed sporogonic development using cultured gametocytes in the Netherlands and naturally circulating strains in Burkina Faso. We quantified the number of sporozoites expelled into artificial skin in relation to intact oocysts, ruptured oocysts, and residual salivary gland sporozoites. In laboratory conditions, higher total sporozoite burden was associated with shorter duration of sporogony (p<0.001). Overall, 53% (116/216) of infected Anopheles stephensi mosquitoes expelled sporozoites into artificial skin with a median of 136 expelled sporozoites (interquartile range [IQR], 34–501). There was a strong positive correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.8; p<0.0001) and a weaker positive correlation between salivary gland sporozoite load and number of sporozoites expelled (ρ = 0.35; p=0.0002). In Burkina Faso, Anopheles coluzzii mosquitoes were infected by natural gametocyte carriers. Among salivary gland sporozoite positive mosquitoes, 89% (33/37) expelled sporozoites with a median of 1035 expelled sporozoites (IQR, 171–2969). Again, we observed a strong correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.9; p<0.0001) and a positive correlation between salivary gland sporozoite load and the number of sporozoites expelled (ρ = 0.7; p<0.0001). Several mosquitoes expelled multiple parasite clones during probing. Whilst sporozoite expelling was regularly observed from mosquitoes with low infection burdens, our findings indicate that mosquito infection burden is positively associated with the number of expelled sporozoites. Future work is required to determine the direct implications of these findings for transmission potential.
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Cyclic nucleotide binding domains (CNB) confer allosteric regulation by cAMP or cGMP to many signaling proteins, including PKA and PKG. PKA of phylogenetically distant Trypanosoma is the first exception as it is cyclic nucleotide-independent and responsive to nucleoside analogues (Bachmaier et al., 2019). Here, we show that natural nucleosides inosine, guanosine and adenosine are nanomolar affinity CNB ligands and activators of PKA orthologs of the important tropical pathogens Trypanosoma brucei, Trypanosoma cruzi, and Leishmania. The sequence and structural determinants of binding affinity, -specificity and kinase activation of PKAR were established by structure-activity relationship (SAR) analysis, co-crystal structures and mutagenesis. Substitution of two to three amino acids in the binding sites is sufficient for conversion of CNB domains from nucleoside to cyclic nucleotide specificity. In addition, a trypanosomatid-specific C-terminal helix (αD) is required for high affinity binding to CNB-B. The αD helix functions as a lid of the binding site that shields ligands from solvent. Selectivity of guanosine for CNB-B and of adenosine for CNB-A results in synergistic kinase activation at low nanomolar concentration. PKA pulldown from rapid lysis establishes guanosine as the predominant ligand in vivo in T. brucei bloodstream forms, whereas guanosine and adenosine seem to synergize in the procyclic developmental stage in the insect vector. We discuss the versatile use of CNB domains in evolution and recruitment of PKA for novel nucleoside-mediated signaling.