Article Figures & Data
Figures
- FIG 1
Deletion of the hp0421 gene perturbs H. pylori cell morphology. (A to C) Confocal microscopy profiles depicting morphological patterns of the Hp76Δ421 (A), Hp76 (B), and Hp76Δ421-reconstituted (C) strains. (D) Scatter plots arraying Hp76wt, Hp76Δ421, and Hp76Δ421-reconstituted cells (recons) based on x-size and y-size analyses performed using CellTool software. (E) CellTool analysis output based on plots representing the distribution of Hp76wt, Hp76Δ421, and Hp76Δ421-reconstituted cell populations (recons) according to the aspect ratio. (F to H) Confocal microscopy images for Hp76Δ421 (F), Hp76 (G), and Hp76Δ421-reconstituted (H) cells when the cells were treated with the filamenting drug aztreonam.
- FIG 2
Flow cytometry analysis of H. pylori cell populations based on their shape. Representative contour plots of H. pylori populations depicted based on flow cytometry analyses. Forward scatter (FSC) plotted on the x axis and side scatter (SSC) on the y axis are presented with mean. (A and B) The Hp26695 strain was grown in the presence and absence of cholesterol for 48 h (A) and 72 h (B). (C and D) The Hp26695 and Hp26695Δ421 strains were grown for 48 h (C) and 72 h (D).
- FIG 3
Absence of cholesteryl glucosides increased H. pylori cell wall permeability. (A and B) Flow cytometry analysis based on influx rates of EtBr as depicted in the form of PE-A histograms delineating the median fluorescence intensity (MFI) for Hp26695 strain grown in the presence and absence of cholesterol (A). (B) Influx rates of EtBr for Hp26695 and Hp26695Δ421 strains grown for 48 h. (C to F) The bars represent MFIs for wild-type, knockout, and reconstituted strains (as annotated) when treated/incubated with EtBr for 5, 10, and 30 min using cultures grown either at 48 h or 72 h (*, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).
- FIG 4
Analysis of LPS expression and biofilm formation. (A) Silver-stained SDS-PAGE gel (15%) depicting profiles of LPS from Hp76, Hp76Δ421, and Hp76Δ421-reconstituted strains. (B) Bar graph representing quantification of biofilm formation by H. pylori after 5 days of incubation. The Hp26695 strain data were analyzed by Student’s t test. The Hp76 strain data were analyzed by one-way ANOVA followed by Tukey’s multiple-comparison tests. (C and D) qRT-PCR analyses of wecA and wzk genes using RNA isolated from wild-type (Hp26695 and Hp76), knockout (Hp26695Δ421 and Hp76Δ421), and Hp76Δ421-reconstituted strains grown for 48 h. (E) Relative mRNA expression of luxS from 2-day-old broth cultures [ns, nonsignificant difference(s); *, P < 0.05, **, P ≤ 0.01; ***, P ≤ 0.001].
Tables
- TABLE 1
MICs of wild-type, Δ421 mutants, and Δ421-reconstituted H. pylori strains
Antibiotic MIC (μg/ml) of the following strain(s)a: Hp26695 Hp26695Δ421 Hp76 Hp76Δ421 Hp76Δ421-
reconstitutedFosfomycin ≥1,024 (R) ≤1.5 ± 0.5 (S) ≥1,024 (R) ≤0.064 ± 2 (S) ≥1,024 (R) Colistin ≥256 (R) ≤12 ± 3 (S) ≥256 (R) ≤10 ± 5 (S) ≥256 (R) Polymyxin B ≥256 (R) ≤8 ± 4 (S) ≥256 (R) ≤10 ± 4 (S) ≥256 (R) Ciprofloxacin ≤0.38 ± 0.5 (R) ≤0.064 ± 0.02 (S) ≤0.50 ± 0.5 (R) ≤0.19 ± 0.2 (S) ≤0.047 ± 0.08 (R) Tetracycline ≤10 ± 3 (R) ≤0.01 ± 0.02 (S) ≤0.50 ± 0.1 (R) ≤0.01 ± 0.09 (S) ≤0.38 ± 0.18 (R) Amoxicillin ≤0.47 ± 0.01 (R) ≤0.016 ± 0.01 (S) ≤0.016 ± 0.01 (S) ≤0.016 ± 0.01 (S) ≤0.016 ± 0.01 (S) Clarithromycin ≤0.001 (S) ≤0.001 (S) ≤0.001 (S) ≤0.001 (S) ≤0.001 (S) ↵a The letters in parentheses after the MICs indicate whether the strain is resistant (R) or sensitive (S) to the respective antibiotic.
- TABLE 2
Primers used in this study
Target gene of the primer Nucleotide sequence of the primer hp0421 US F GTGGATTATGACTCTTTAGAGACTTG hp0421 US R GTGCCATGGCTCGAGTTAACTACTCTTCTTTAAAATTGAAT hp0421 DS F GTGCCATGGCTCGAGTGAAAGGATAAAAAATGCAAGAA hp0421 DS R CCAATTTTAGGGCAGGCTAAAAAC wecA F ATGGTGCTTGGGTTTATGGTG wecA R GGCTTTCTGGCGTTTTATTTTG wzk F AAACTCAAAGACAACCACGAAG wzk R CGACCGCTAAAATCAACAAG 16s rRNA F GGTAAAATCCGTAGAGATCAAGAGG 16s rRNA R ACAACTAGCATCCATCGTTTAGG cds1 F GGATGAATTTTTAGACGATTTGC cds1 R CCCTCTTCTTTTTCTTCTTCAGG cds3 F CTAAACATGGCAGCTTGATCC cds3 R AATGGATTTCAACCACCTTCC luxS F TTTGATTGTCAAATACGATGTGC luxS R TGTGAGATAAAATCCCGTTTGG
FIG S1
Morphology of H. pylori in the absence of cholesterol in culture and upon deletion of hp0421. (A and B) Confocal microscopy images depicting H. pylori grown in the absence (A) and presence (B) of cholesterol. (C) Scatter plots showing Hp26695 and Hp26695Δ421 cell populations by x size and y size as analyzed by CellTool. (D) Graphical profiles representative of the distribution of Hp26695 and Hp26695Δ421 cell populations according to the aspect ratio and as analyzed by CellTool are shown. (E and F) RNA isolated from and the relative mRNA expression analyses of log-phase cultures of Hp26695 and Hp76 strains as quantified by qRT-PCR of csd1 and csd3 gene loci (ns denotes nonsignificant differences at P ≥ 0.05). Download FIG S1, TIF file, 2.1 MB.
Copyright © 2018 Qaria et al.
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FIG S2
Morphology of Hp76 strains presented by flow cytometry. Representative contour plots of H. pylori populations when analyzed by flow cytometry; FSC and SSC are shown on the x and y axes, respectively, with mean and median. Hp76Δ421 displayed high FSC and SSC compared to the Hp76 and Hp76Δ421-reconstituted strains grown for 48 h (A) and 72 h (B). Download FIG S2, TIF file, 0.5 MB.
Copyright © 2018 Qaria et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S3
The absence of cholesteryl glucosides caused increased cell wall permeability. (A to D) PE-A histograms delineating the influx rates of EtBr represented as MFI for different wild-type and knockout strains grown in culture for different periods and when treated with EtBr at different time points as shown. Download FIG S3, TIF file, 1.1 MB.
Copyright © 2018 Qaria et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
