Article Figures & Data
Figures
- FIG. 1.
PapX is transcribed as part of the pap operon. (A) Schematic of the papG and papX genes of CFT073. papX is 263 bp downstream of the papG allele. Arrows indicate primer sequences designed to amplify a 482-bp product (papG-papX). (B) RT-PCR using papG-papX primers (top), as well as control primers within the papA gene (bottom), to determine whether papX is transcribed with papG. CFT073 genomic DNA (wild-type DNA) and water are included as positive and negative controls. A “+” or “-” indicates the presence or absence of reverse transcriptase in the cDNA synthesis reaction.
- FIG. 2.
Overexpression of papX in UPEC CFT073 represses motility and flagellar production. (A) In vitro growth curves of CFT073(pLX3607) (vector control; ▪) and CFT073(pDRM001) (encodes papX; □) with (red) or without (black) addition of 0.5 mM IPTG. (B and C) The motilities of CFT073(pLX3607) (B) and CFT073(pDRM001) (C) in soft agar without IPTG are shown. White bars indicate the diameter of motility of each strain. (D and E) Indirect immunofluorescence analysis of flagellum expression. Suspensions of CFT073(pLX3607) (D) and CFT073(pDRM001) (E) were spotted onto microscope slides and stained with antibody specific to H1 flagellin of CFT073 (green). Bacterial nucleic acids were visualized by staining with propidium iodide (red). Scale bars, 10 μm. (F) Western blot analysis of flagellin production. Whole-cell lysates from standardized mid-log-phase cultures were prepared, electrophoresed onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and subjected to Western blot analysis with antiserum specific to H1 flagella. The molecular mass marker is indicated on the left in kilodaltons.
- FIG. 3.
qPCR analysis of expression of selected UPEC genes identified during microarray analysis. Black bars represent the average fold changes (n = 3) in gene expression. (A) qPCR results for CFT073 ΔpapX. Changes were calculated using wild-type CFT073 as the relative measure of comparison. (B) qPCR results for CFT073(pDRM001) encoding papX. Changes were calculated using CFT073 carrying the vector plasmid pLX3607 alone as the relative measure of comparison. Error bars represent the standard errors of the means.
- FIG. 4.
Binding of PapX to the promoter of flhD as revealed by EMSA. All lanes contained 0.2 ng of either a DIG-labeled 500-bp DNA fragment corresponding to the flhD promoter (left panel) or a DIG-labeled 300-bp DNA fragment containing the gapA promoter (right panel). Increasing amounts of purified PapX protein were mixed with the DIG-labeled probes as indicated. A 100-fold excess of unlabeled flhD promoter DNA was included (left panel) as a control to illustrate the specificity of the PapX-flhD binding reaction.
- FIG. 5.
Cochallenges of mice with UPEC CFT073 and CFT073 ΔpapX. In three separate experiments, a total of 30 mice were transurethrally inoculated with a 1:1 mixture of the wild type and the mutant. At 48 h postinfection, mice were sacrificed and bacterial counts in the bladder and kidneys were determined. Each data point is represents the log10 CFU per gram of tissue collected per mouse. Bars indicate the median log10 CFU per gram of tissue collected for each group. The limit of detection of this assay is 102 CFU/g tissue. Statistical differences between the median number of log10 CFU per ml or per gram tissue for UPEC CFT073 and its ΔpapX mutant were determined by using the Wilcoxon rank sum test. P values are indicated below the graph.
Tables
- TABLE 1.
Primers used in this study
Analysis and primer Sequence (5′ to 3′) qPCR fliC F ACAGCCTCTCGCTGATCACTCAAA fliC R GCGCTGTTAATACGCAAGCCAGAA flhD F TCCGCTATGTTTCGTCTCGGCATA flhD R ACCAGTTGATTGGTTTCTGCCAGC flhC F AAACTGGCTTGTGTAATGGCGTCG flhC R TCAACAAACCGCACCAATGTCCAG fliA F AACGCTATGACGCCCTACAAGGAA fliA R AGTTCCTGCTCCAGTTGCCCTATT yhjH F TATTAACGGTGGTCACGCATCCCT yhjH R TGCTCTTTCACAACCTCCATCCGA sat F ATGGATTCAGGCTGAGTGGTGGAA sat R TTCAGTTTCAGGCTGTCCCAGAGT lrhA F AATGCTGGAATCCCAGGAAGTGGA lrhA R ACCATATCGCGAAACGGACTTGGA sfaB/focB F TCCGGCTCCATGTCTGAAGAACAA sfaB/focB R TTACGGGAATGACCACTGACCAGA gapA F CGTTAAAGGCGCTAACTTCG gapA R ACGGTGGTCATCAGACCTTC His-tagged PapX PapX-His F CATATGCGCGCTTGTACACAGACA PapX-His R GGTACCTGAGCTGACATCATCAAGAT EMSA flhD F GTTGTGCGGTAAGTGTTTG flhD R CCAGAATAACCAACT gapA F ACGTGACTGATTCTA gapA R CAGCTATTTGTTAGTG fliA F AACTGAGACTGACGGCAAC fliA R GATAAACAGCCCTGCGTTA fliC F TCGACACGTAAAACGAATACC fliC R GATTCGTTATCCTATATTGC - TABLE 2.
Flagellar and motility genes upregulated in CFT073 ΔpapX
Gene Annotation Fold change in CFT073 ΔpapXa flhD Flagellar transcriptional activator 1.74 flhC Flagellar transcriptional activator 1.96 fliL Flagellar biosynthesis protein 1.8 fliE Flagellar hook-basal body complex protein 2.16 flgB Flagellar basal body rod protein 1.78 flgC Flagellar basal body rod protein 1.49 flgE Flagellar hook protein 1.64 flgF Flagellar basal body rod protein 1.79 flgG Flagellar basal body rod protein 1.64 flgH Flagellar L-ring protein precursor 1.79 flgI Flagellar P-ring protein precursor 2.04 fliA RNA polymerase sigma factor (σ28) 1.52 flgA Flagellar basal body P-ring formation protein 1.59 aer Aerotaxis receptor 2.55 yhjH EAL domain-containing protein involved in flagellar function 1.97 ↵ a Compared to expression levels in wild-type CFT073. P < 0.005.
- TABLE 3.
Flagellar and motility genes downregulated in CFT073 overexpressing papX
Gene Annotation Fold change in CFT073 (pDRM001)a flhD Flagellar transcriptional activator -1.82 flhC Flagellar transcriptional activator -1.98 fliH Flagellar biosynthesis protein -2.31 fliJ Flagellar biosynthesis protein -1.79 fliK Flagellar hook length control protein -2.10 fliL Flagellar biosynthesis protein -2.53 fliM Flagellar motor switch protein -2.14 fliE Flagellar hook-basal body complex protein -1.88 flhB Flagellar biosynthesis protein -1.57 flgB Flagellar basal body rod protein -2.39 flgC Flagellar basal body rod protein -2.11 flgD Flagellar basal body rod modification protein -1.87 flgE Flagellar hook protein -2.20 flgF Flagellar basal body rod protein -1.85 flgG Flagellar basal body rod protein -2.01 flgH Flagellar L-ring protein precursor -1.82 flgJ Peptidoglycan hydrolase -1.80 fliA RNA polymerase sigma factor (σ28) -2.27 fliZ Predicted regulator of FliA activity -2.00 flgA Flagellar basal body P-ring formation protein -1.91 flgM Negative regulator of flagellar synthesis (anti-σ28) -2.08 flgK Flagellar hook-associated protein 1 -1.91 flgL Flagellar hook-associated protein 3 -2.04 fliD Flagellar hook-associated protein 2 -3.89 fliS Flagellar protein FliS -2.41 fliC Flagellin -2.20 motA Chemotaxis protein -2.50 motB Chemotaxis protein -2.87 cheA Chemotaxis protein -3.43 cheW Chemotaxis protein -3.01 tar Methyl-accepting chemotaxis protein II -2.57 tsr Methyl-accepting chemotaxis protein I -2.89 cheR Chemotaxis protein methyltransferase -1.87 cheB Protein-glutamate methylesterase -1.92 cheY Chemotaxis protein -3.05 cheZ Chemotaxis protein -2.60 aer Aerotaxis receptor -2.46 yhjH EAL domain containing protein involved in flagellar function -3.46 ↵ a pDRM001 encodes papX. Values are given compared to the expression levels in the vector control CFT073(pLX3607). P < 0.005.
- TABLE 4.
Non-motility-associated genes differentially expressed in CFT073 ΔpapX and CFT073 overexpressing papX
Gene Annotation Fold changea in: CFT073 ΔpapXb CFT073 (pDRM001)c papX PapX protein -2.07 11.00 ibpA Heat shock protein A -2.62 sat Secreted autotransporter toxin 2.01 sfaB/focB Putative F1C and S fimbrial switch regulatory protein 2.73 ybcL Putative kinase inhibitor 1.50 ymdA Hypothetical protein 3.32 -1.74 ydjR Hypothetical protein 1.55 -1.75 c3568 Hypothetical protein -1.56 c3190 Hypothetical protein 2.66 c3189 Hypothetical protein 1.75 c3188 Hypothetical protein 1.74
