Targeting plasmid-encoded proteins that contain immunoglobulin-like domains to combat antimicrobial resistance
- Department of Genetics, Microbiology and Statistics, University of Barcelona, Spain
- Department of Biochemistry and Physiology, Universitat de Barcelona, Spain
- Institut de Nutrició i Seguretat Alimentària, Universitat de Barcelona, Spain
- Department of Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CNB-CSIC), Spain
- Instituto Universitario de Investigaciones Biomédicas y Sanitarias (IUIBS), Facultad de Veterinaria, Universidad de Las Palmas de Gran Canaria (ULPGC), Campus Universitario de Arucas, Spain
- Institute for Bioengineering of Catalonia, The Barcelona Institute of Science and Technology, Spain
Figures
Figure 1
Infection of mice with the S. Typhimurium SL1344 and S. Typhimurium (pHCM1) strains.
Survival rate (A), body weight evolution (B), disease index (C), and proinflammatory cytokine expression in spleen, Il-6 (D), Il-1β (E), and Tnf-α (F). Groups treated with ampicillin (Amp) are indicated with + Amp. In panel (A), the survival rate of control animals and those infected with the S. Typhimurium SL1344 (WT) strain and treated with Amp, are 100%. Animals infected with the S. Typhimurium (pHCM1) strain have the same survival rate independently of the treatment with Amp. Results are expressed as mean ± SEM (n=6–8 animals). Means without a common letter differ, p<0.05. The survival rates were compared by Log-rank (Mantel-Cox) test and expressed as the percentage of survival. Body weight evolution was analyzed by means of repeated measures ANOVA. Disease indices are expressed as median values and quartiles, and were compared by the Kruskal-Wallis test. The expression of cytokines were analyzed by using one-way analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD) post hoc test.
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Figure 1—source data 1
The Excel file containing numerical source data of the results is shown in Figure 1.
- https://cdn.elifesciences.org/articles/95328/elife-95328-fig1-data1-v1.xlsx
Figure 2
Effect of immunization with the RSP protein on body weight evolution (A) and immunoglobulin concentration (B–I).
Grey symbols represent non-immunized (No-Imm) mice; black symbols represent immunized (Imm) mice. Results are expressed as mean ± SEM (n=6–8 animals). IgA, immunoglobulin A; IgG, immunoglobulin G; nd, non-detected. Means without a common letter differ, p<0.05. Data were analyzed with Student’s t-test.
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Figure 2—source data 1
The Excel file containing numerical source data of the results is shown in Figure 2.
- https://cdn.elifesciences.org/articles/95328/elife-95328-fig2-data1-v1.xlsx
Figure 3
Survival rate (A), body weight change (B), and clinical signs (C) after challenge with the SL1344 (pHCM1) strain.
Grey symbols represent non-immunized (No-Imm) mice; black symbols represent immunized (Imm) mice. In panel B, results are expressed as mean ± SEM (n=12–14 animals). Means without a common letter differ, p<0.05. The survival rates were compared by Log-rank (Mantel-Cox) test and expressed as the percentage of survival. Disease indices are expressed as median values and quartiles, and were compared by the Kruskal-Wallis test.
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Figure 3—source data 1
The Excel file containing numerical source data of the results is shown in Figure 3.
- https://cdn.elifesciences.org/articles/95328/elife-95328-fig3-data1-v1.xlsx
Figure 4
Immunoglobulin concentration and cytokine expression in the gastrointestinal tract after RSP immunization and challenge with Salmonella.
Total IgA concentration in jejunum content (A), in colon content (B), and in feces (C). RSP-specific IgA titers in jejunum content (D), in colon content (E), and in feces (F). Il-1β (G), Il-6 (H), and Tnf-α (I) expression in colon mucosa. Open bars represent non-immunized (Non-Imm) mice; solid bars represent immunized (Imm) mice. Results are expressed as means ± SEMs (n=10–12 animals). Means without a common letter differ, p<0.05. IgA, immunoglobulin A; IgG, immunoglobulin G; Il, interleukin; Int, the interaction between both factors; Tnf-α, tumor necrosis factor-alpha. All data were analyzed using two-way ANOVA (Immunization and infection factors).
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Figure 4—source data 1
The Excel file containing numerical source data of the results is shown in Figure 4.
- https://cdn.elifesciences.org/articles/95328/elife-95328-fig4-data1-v1.xlsx
Figure 5
Immunoglobulin concentration, spleen weight, Salmonella counts in spleen tissue, and cytokine expression in spleen tissue after immunization with the RSP protein and challenge with the SL1344 (pHCM1) strain.
Total IgG concentration (A) and RSP-specific IgG titers (B) in plasma. Spleen weight (C) and Salmonella counts in spleen tissue (D). Il-1β (E), Il-6 (F) and Tnf-α (G) expression in spleen tissue. Results are expressed as means ± SEMs (n=12–14 animals). Means without a common letter differ, p<0.05. IgA, immunoglobulin A; IgG, immunoglobulin G; Il, interleukin; nd, non-detected; Tnf-α, tumor necrosis factor-alpha. All data were analyzed using two-way ANOVA (Immunization and infection factors).
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Figure 5—source data 1
The Excel file containing numerical source data of the results is shown in Figure 5.
- https://cdn.elifesciences.org/articles/95328/elife-95328-fig5-data1-v1.xlsx
Figure 6 with 1 supplement
Selection of RSP-binding Nbs from immune libraries displayed on the surface of E. coli.
(A) Flow cytometry analysis of the enrichment of bacterial population displaying Nb libraries generated after the immunization of dromedaries with the C-terminal domain of the RSP protein. Three rounds of selection with the RSP protein by magnetic cell sorting (MACS) and one round of fluorescence-activated cell sorting (FACS) were performed, in which bacteria were incubated with 50 nM of biotin-labeled RSP (or 100 nM of fibrinogen used as a specificity control) and stained with Streptavidin-APC. (B) Flow cytometry of the individual bacterial clones selected from the immune library. The bacterial surface display of the corresponding nanobody was detected using an anti-c-myc monoclonal antibody. The binding of biotin-labeled antigens to Nbs displayed on bacteria was performed with the incubation of bacterial cells with 50 nM of biotin-labeled RSP.
Figure 6—figure supplement 1
Amino acid sequence alignment of the selected VHHs after the magnetic cell sorting (MACS) and fluorescence-assisted cell sorting (FACS) enrichment protocol of the bacterial library generated after the immunization of two dromedaries with the RSP protein.
Red color, blue color, and purple color show the CDR1, CDR2, and CDR3 of the corresponding VHH, respectively. Yellow color marks the sequence of the c-myc tag used to evaluate the Nb display on the bacterial surface by flow cytometry. Alignment generated with Clustal Omega. Labels indicate full level of conservation (*) or decreased degree of conservation (: or.).
Figure 7
Analysis of the interaction of the VHH-RSP#3 isolated in the immune library with the native RSP protein at a macro and microscopic level.
(A) Assay of the ability of E. coli DH10BT1R cells expressing nanobodies on its surface to agglutinate cells of the S. Typhimurium SL1344 (R27) strain. Blue arrow marks the aggregation of bacteria after the interaction of E. coli cells producing nanobodies directed against the RSP protein (VHH-RSP #3) and Salmonella cells harboring the R27 plasmid. (B) Transmission electron microscopy imaging mixtures of E. coli DH10BT1R cells expressing nanobodies against the RSP protein on its surface and cells of the S. Typhimurium SL1344 strain, harboring or not the R27 plasmid. The studies were performed by labeling the nanobodies-producing bacteria (E. coli DH10BT1R) with a mouse antibody anti c-myc-tag and goat anti-mouse IgG conjugated to 12 nm gold particles. Blue bars represent 1 μm while black bars represent 2 μm.
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Figure 7—source data 1
Full-length images of transmission electron microscopy pictures shown in Figure 7B.
- https://cdn.elifesciences.org/articles/95328/elife-95328-fig7-data1-v1.zip
Figure 8
Characterization of the purified VHH-RSP#3 as a specific binder to the RSP protein.
(A) Purification of Nb3-Fc by affinity column. Coomassie staining of the SDS-PAGE (10%) showing the purified Nb-RSP #3-Fc from transfected mammalian cell culture supernatants. Molecular weight markers are indicated on the left. (B) ELISA shows the binding capacity of the purified VHH-RSP#3-Fc to the RSP or BSA protein. The plot represents the OD values at 490 nm obtained with the indicated concentrations of the purified Nb3-Fc after 1 hr of interaction with the corresponding antigen.
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Figure 8—source data 1
Original file for the Coomassie staining of the SDS-PAGE in Figure 8A.
- https://cdn.elifesciences.org/articles/95328/elife-95328-fig8-data1-v1.zip
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Figure 8—source data 2
PDF containing Figure 8A showing the Coomassie staining of the SDS-PAGE with highlighted sample labels corresponding to the purified Nb-RSP #3-Fc.
- https://cdn.elifesciences.org/articles/95328/elife-95328-fig8-data2-v1.pdf
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Figure 8—source data 3
The Excel file containing numerical source data of the results is shown in Figure 8B.
- https://cdn.elifesciences.org/articles/95328/elife-95328-fig8-data3-v1.xlsx
Figure 9
Interference of E. coli DH10BT1R cells expressing nanobodies on its surface with the conjugative transfer of the R27 plasmid in Salmonella.
R27 plasmid was conjugated from a donor strain (SL1344) to a recipient strain (SL1344 ibpA::lacZ-Kmr) with the absence (labeled as ‘none’) or presence of the interferent strains E. coli DH10BT1R, plasmid-free clone; E. coli DH10BT1R (pNVFib1), clone expressing a fibrinogen-specific VHH; E. coli DH10BT1R (pNeae2 VHH-RSP #3), clone expressing RSP-specific VHH. Results are expressed as means ± SEMs (n=4 independent biological replicates). Means without a common letter differ, p<0.05. Data were analyzed with Student’s t-test.
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Figure 9—source data 1
The Excel file containing numerical source data of the results is shown in Figure 9.
- https://cdn.elifesciences.org/articles/95328/elife-95328-fig9-data1-v1.xlsx
Additional files
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Supplementary file 1
Strains, plasmids and oligonucleotides used in this work.
(a) List of the strains and plasmids used in this work. (b) List of the oligonucleotides used in this work.
- https://cdn.elifesciences.org/articles/95328/elife-95328-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/95328/elife-95328-mdarchecklist1-v1.docx
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Targeting plasmid-encoded proteins that contain immunoglobulin-like domains to combat antimicrobial resistance
eLife 13:RP95328.
https://doi.org/10.7554/eLife.95328.3
