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Proteomic analysis of the host–pathogen interface in experimental cholera

Nature Chemical Biology volume 17, pages 1199–1208 (2021)Cite this article

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Abstract

The microbial cell surface is a site of critical microbe–host interactions that often control infection outcomes. Defining the set of host proteins present at this interface has been challenging. Here we used a surface-biotinylation approach coupled to quantitative mass spectrometry to identify and quantify both bacterial and host proteins present on the surface of diarrheal fluid-derived Vibrio cholerae in an infant rabbit model of cholera. The V. cholerae surface was coated with numerous host proteins, whose abundance were driven by the presence of cholera toxin, including the C-type lectin SP-D. Mice lacking SP-D had enhanced V. cholerae intestinal colonization, and SP-D production shaped both host and pathogen transcriptomes. Additional host proteins (AnxA1, LPO and ZAG) that bound V. cholerae were also found to recognize distinct taxa of the murine intestinal microbiota, suggesting that these host factors may play roles in intestinal homeostasis in addition to host defense.

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Fig. 1: The diarrheal fluid proteome in infant rabbits is largely stimulated by CT.
Fig. 2: Identification of surface-exposed V. cholerae proteins and V. cholerae-bound host-derived proteins.
Fig. 3: SP-D associates with V. cholerae cells in diarrheal fluid and in vitro.
Fig. 4: SP-D is an intestinal mucosal defense factor.
Fig. 5: LPO, AnxA1 and ZAG interact with V. cholerae.
Fig. 6: HBBPs interact with gut commensal bacteria.

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Data availability

All data, reagents and strains presented in this study are reported in the paper and associated Supplementary Information. Proteomic datasets were deposited to the ProteomeXchange Consortium via PRIDE (Data set identifier PXD027076 and 10.6019/PXD027076). RNA-seq datasets were deposited to the NCBI GEO repository (data set identifier GSE179530). Source data are provided with this paper.

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Acknowledgements

We thank members of the Waldor laboratory for helpful discussions, M. Chao for insightful comments on the manuscript, R. Rodrigues, A. Warr and Y. Hasegawa for expert help with proteomics and bioinformatics and the Bettencourt-Schueller foundation for support. Glycomic experiments were done with the participation of the Protein-Glycan Interaction Resource of the Center for Functional Glycomics, and the National Center for Functional Glycomics, supporting grant nos. P41 GM103694 and R24 GM137763. Work in M.K.W. laboratory is supported by HHMI and National Insitutes of Health grant no. R01 AI-042347. T.Z. was supported by a Sarah Elizabeth O’Brien Trust Postdoctoral Fellowship. A.Z. was supported by an EMBO long-term fellowship (ALTF 1514-2016) and by a HHMI Fellowship of the Life Sciences Research Foundation.

Author information

Authors and Affiliations

  1. Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, MA, USA

    Abdelrahim Zoued, Hailong Zhang, Ting Zhang, Carole J. Kuehl, Bolutife Fakoya, Brandon Sit & Matthew K. Waldor

  2. Department of Microbiology, Harvard Medical School, Boston, MA, USA

    Abdelrahim Zoued, Hailong Zhang, Ting Zhang, Rachel T. Giorgio, Carole J. Kuehl, Bolutife Fakoya, Brandon Sit & Matthew K. Waldor

  3. Howard Hughes Medical Institute, Boston, MA, USA

    Abdelrahim Zoued, Hailong Zhang, Carole J. Kuehl & Matthew K. Waldor

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Contributions

A.Z. and M.K.W. conceived and designed the study. A.Z., H.Z., T.Z., B.F. and C.J.K, performed all experiments. A.Z. and R.T.G. analyzed data. A.Z., B.S. and M.K.W. wrote the manuscript and all authors edited the paper.

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Correspondence to Matthew K. Waldor.

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Extended data

Extended Data Fig. 1 Diarrheal fluid volume and composition.

(a) Schematic of the experimental protocol for identification of the proteome in diarrheal fluid isolated from rabbits inoculated with V. cholerae, V. cholerae Δctx, purified cholera toxin (CT) or buffer alone (Mock). (b) Bacterial burdens recovered from diarrheal fluid harvested from rabbits infected with V. cholerae and V. cholerae Δctx. Data are mean for 3 independent animals per group. (c) Diarrheal fluid volumes collected from rabbits infected with V. cholerae, V. cholerae Δctx, purified cholera toxin (CT) and buffer (Mock). Data are mean for 3 independent animals for V. cholerae, V. cholerae Δctx and mock, and 4 independent animals for CT-infected animals. (d) Predicted localization of rabbit proteins identified in diarrheal fluid. Bioinformatic analysis was performed using the G:Profiler (http://biit.cs.ut.ee/gprofiler/) webtool. (e) Scatterplot of relative fold changes in protein abundances isolated from rabbit infected with V. cholerae Δctx (Delta) compared to wild-type V. cholerae (Vc), each relative to the proteomes of mock infected animals. The red dot indicates SP-D.

Extended Data Fig. 2 Validation of the surface biotinylation assay.

(a) Controls validating the surface-biotinylation screen. (a) Proteins isolated following surface-biotinylation protocol with (+) or without (-) the biotinylation step were separated by 10% acrylamide SDS–PAGE and silver-stained. (b) Presence of cytoplasmic RNA polymerase β and (c) outer-membrane OmpU were assessed by immunostaining with anti-RNApol and anti-OmpU antibodies, respectively. T: total V. cholerae lysate. Western blot analyses were performed at least three times with consistent results. (d) Venn diagram showing the comparison of V. cholerae proteins identified with our surface-biotinylation screen and V. cholerae outer membrane vesicles (OMV’s) proteomes from (34); 181 and 110 are the total number of proteins from each group.

Source data

Extended Data Fig. 3 SP-D associates with V. cholerae cells.

(a,b) Immunofluorescence micrographs of rabbit small intestines inoculated with V. cholerae-GFP. Bacterial cells were detected by GFP fluorescence, SP-D was detected with a goat anti-SP-D antibody followed by anti-goat antibody coupled to Alexa fluor 468. Phalloidin (for actin labeling) is stained with an antibody coupled to Alexa fluor 647 and DAPI (for DNA labeling) is shown is blue. (b) Only anti-goat antibody coupled to Alexa fluor 468 was used to assess non-specific staining of the second antibody. Scale bar is 100 μm. (c) V. cholerae cells were incubated in PBS containing 5 mM CaCl2 for 1 hour at 37 °C in the presence of SP-D (10 μg/ml), SP-D ΔC-ter (10 μg/ml) or a denatured SP-D and then incubated with fluorescent nucleic acid stains SYTO 9 and Propidium Iodide to assess viability. Data are mean ± s.d for three biological replicates. (d) Wide field of micrographs shown in Fig. 3e are represented by the dotted white square. Additional fields are also shown in rows #2 and #3. Scale bar is 10 μm. (e) Degradation of SP-D over time upon incubation with V. cholerae C6706 or the protease deficient strain V. cholerae C6706 Δquad. Graph shows SP-D protein band intensity normalized to RNApolβ over time. Western blot analyses were performed at least three times with consistent results.

Source data

Extended Data Fig. 4 Transcriptomic analysis.

(a) mRNA levels of the SFTPD gene measured by qRT-PCR and normalized to GAPDH mRNA levels in arbritrary unit (A.U) in mice infected with V. cholerae, V. cholerae Δctx or mock. Data are shown as mean ± s.d. (n = 4 technical replicates). (b) V. cholerae Δctx small intestinal colonization in littermate sftpd-/- and sftpd +/+ mice. Bacterial burdens recovered from proximal and distal small intestine 18 hrs after V. cholerae Δctx inoculation. Data are mean for 12 and 7 independent SP-D+ and SP-D- animals respectively. Note S-PD + include both heterozygotes (sftpd +/−) and homozygous (sftpd +/+) animals. Statistical significance was determined using a Mann-Whitney U t test. (c) Principal Component Analysis (PCA) plot of RNA-seq data from four biological replicates of WT or sftpd-/- mice proximal small intestine infected with V. cholerae. (d) Principal Component Analysis (PCA) plot of RNA-seq data from four biological replicates of WT or sftpd-/- mice distal small intestine infected with V. cholerae. (e) Heat map of rlog-transformed read counts for 4 animal replicates (WT or sftpd-/- infected with V. cholerae) for top 30 and bottom 30 genes by rank.

Extended Data Fig. 5 LPO, AnxA1 and ZAG binding to microbial glycans.

(a-c) Results of ZAG (a), LPO (b) and AnxA1 (c) binding to Microbial Glycan Microarray organized by genus and species (red is 5 and blue is 50 μg ml−1). Data are presented as the mean ± s.d. (n = 4 of a technical replicate for each immobilized glycan). Note: scales on Y axes are different. The complete datasets are available in Supplementary Data Set 4. (d) AnxA1-lipid interaction assessed by protein-lipid overlay assays. Graph shows quantification of the Annexin A1 protein band intensity normalized to the intensity of a blank spot band. Lysophosphatidic acid (LPA), Lysophosphocholine (LPC), Phosphatidylinositol (PtdIns), Phosphatidylinositol(3)-phosphate (PtdIns(3)P), Phosphatidylinositol (4)-phosphate (PtdIns(4)P), Phosphatidylinositol (5)-phosphate (PtdIns(5)P), Phosphatidylethanolamine (PE), Phosphatidylcholine (PC), Sphingosine-1-Phosphate (S1P), Phosphatidylinositol(3,4)-bisphosphate (PtdIns(3,4)P2), Phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P2), Phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P2), Phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3), Phosphatidic acid (PA), Phosphatidylserine (PS).

Extended Data Fig. 6 Microbiota-bound 16S analysis.

(a) Representative gating strategy illustrating bacterial populations coated with HBBP. (b) Principle component analyzes based on the Bray Curtis β-diversity metric showing that samples of AnxA1, LPO and ZAG positive populations each form separate clusters whereas all the HBBP negative populations cluster together. (c) Alpha rarefaction plot. Shown are the number of different observed features as a function of the number of sequences analyzed and generated with QIIME2. (d) Relative abundance differences between bound and unbound fractions of the gut symbionts, taxa with significant p values are shown as red circles (two-sided Welch’s t statistical as implemented in aldex2). clr: center log-ratio; f: family; g: genus.

Supplementary information

Reporting Summary

Supplementary Table 1

Complete intestinal fluid proteomes.

Supplementary Table 2

Complete pathway analysis.

Supplementary Table 3

Full proteomic datasets.

Supplementary Table 4

Transcriptomic analysis.

Supplementary Table 5

Full glycomic analysis.

Supplementary Table 6

List of strains.

Supplementary Table 7

List of primers.

Supplementary Table 8

List of antibodies.

Source data

Source Data Fig. 3

Unprocessed western blot.

Source Data Fig. 5

Unprocessed western blot.

Source Data Extended Data Fig. 2

Unprocessed western blot.

Source Data Extended Data Fig. 3

Unprocessed western blot.

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Zoued, A., Zhang, H., Zhang, T. et al. Proteomic analysis of the host–pathogen interface in experimental cholera. Nat Chem Biol 17, 1199–1208 (2021). https://doi.org/10.1038/s41589-021-00894-4

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