Article Figures & Data
Figures
- FIG. 1.
Schematic representation of a segment of pRAP containing rpeA. The white arrow represents rpeA (3,684 bp), and the dark arrows represent other open reading frames carried by this fragment. HP is hypothetical protein. The domain organization of RpeA is indicated below the fragment and shows the N-terminal cleavage site predicted by the SignalP method and two N-terminal conserved motifs as well as the serine protease motif, the proline-rich tandem repeat in the passenger domain, and the conserved YEF motif at the C terminus. The predicted molecular mass of each domain is indicated in kDa.
- FIG. 2.
Analysis of ral and rpeA expression by RT-PCR in wild-type REPEC strains E22 (left panels) and 83/39 (right panels). (A) Expression analysis of rpeA using primers rpeA-F and rpeA-R, internal to rpeA. (B) Expression of ralG-ralH using primers ralG-F and ralH-R. (C) Expression analysis of ralI-rpeA using primers ralI-F and rpeA-R, yielding a 1,353-bp product. (D) Expression analysis of ralI-rpeA using primers ralI-F and rpeA-RT, yielding a 920-bp product. Std indicates DNA size markers shown in base pairs; DNA indicates the PCR control; RT− indicates RT-PCR in the absence of reverse transcriptase; RT+ indicates PCR from cDNA.
- FIG. 3.
Quantitative adherence of E. coli XL1-Blue carrying pRpeA (white bars) and E. coli XL1-Blue carrying pUC18 (black bars) to HeLa and HCT-8 cells. Results are expressed as percentages of the original inoculum recovered from quadruplicate wells and are the means for at least four independent experiments. *, adherence significantly greater than that of E. coli XL1-Blue carrying pUC18 (P < 0.05; unpaired, two-tailed t test).
- FIG. 4.
Mean weights (circles) and mean log10 numbers of CFU per swab (triangles) of rabbits infected with wild-type REPEC 83/39 (solid shapes) and the REPEC ΔrpeA mutant (open shapes). Animals were weighed daily, and fecal swabs were taken daily for 27 days after inoculation. The dotted line indicates the CFU detection limit.
Tables
- TABLE 1.
Oligonucleotide primers used in this study
Primer Nucleotide sequencea P2 5′-TACCTACAACCTCAAGCT-3′ STM-R2 5′-CAAACCGTTATTCATTCG-3′ rpeA-F 5′-CTTACACGTGGCACTAG-3′ rpeA-R 5′-CAGTGATCGCTGAAGGAG-3′ ral-F 5′-ATGGATAAGCGTACGCC-3′ ral-R 5′-AATACTAACTGGTAGGG-3′ Tn5F 5′-CAGGCATGCAAGCTTCG-3′ Tn5R 5′-GGTACCGAGCTCGAATT-3′ prpeA-F 5′-CGGGATCCCGATCTCGAACAACATTTACACG-3′ prpeA-R 5′-CGGGATCCCGTTACTTGTCGTCATCGTCTTTGTAGTCGAACTCATATTTCAAACCAAG-3′ pKD4rpeA-F 5′-ATGAACAAGATTTTTTCACTTAAATTTAGCACCGCTTCTGGTGGTTTAATTGCATGTGTAGGCTGGAGCTGCTTCG-3′ pKD4rpeA-R 5′-TCAGAACTCATATTTCAAACCAAGAATTCCAACAGTATCGGTTTTGAAGTCACCACCATATGAATATCCTCCTTA-3′ ralI-F 5′-CTGGTACTGGGAAATTAC-3′ rpeA-RT 5′-GAAGCAATATGGCGTCG-3′ ralG-F 5′-CAAATTCAGTTATTGGTAC-3′ ralH-R 5′-CCATTTTGGAGCCTCATC-3′ ↵ a Engineered restriction sites are underlined, and pKD4 sequences are italicized.
- TABLE 2.
Transposon insertion sites of REPEC 83/39 mutants attenuated for colonization
Group and strain(s) Disrupted gene or homologue Putative functiona LEE 83E7 escV T3SS 81F9 escD T3SS 54F5 rorf8 T3SS 81E6 map T3SS effector 28A7 espB T3SS translocator/effector 91A6 tir T3SS effector Fimbriae 81F5 ralD Fimbrial usher 91H5 ralH Minor fimbrial subunit 81H9 ralI Minor fimbrial subunit 28F5 z2203 Fimbrial usher Cell wall 28E1 yaiP Glycosyltransferase 91F9 waaD Lipopolysaccharide synthesis 91H9 z1190 Glycosyltransferase 54F9 nlpA Predicted lipoprotein 67F12 nlpC Predicted lipoprotein Regulation 81A8 afrR Transcriptional activator 91E1 z3632 Sensor histidine kinase similar to EvgA Metabolism 80E1 ydiF Acetoacetyl-coenzyme A transferase 67A6 cadB Lysine/cadaverine antiporter 91A10 lysP Lysine specific permease 54A6 gltB Glutamate synthase Other 54H9 z0853 Hypothetical protein 67F5 ydfQ Hypothetical protein 91F7, 28A3 z1419, z1420 Hypothetical proteins 28H2 b1168 Putative membrane protein 81H5 rpeA Putative autotransporter ↵ a T3SS, type III secretion system.
- TABLE 3.
Shared amino acid sequence similarity between RpeA and its closest relatives
Protein GenBank accession no. Major reported function Length (amino acids) % Amino acid identity for: Entire protein Leader sequence Passenger domain β domain Hbp/Tsh CAA11505 Hemoglobin binding protein/hemagglutinin 1,377 37 48 39 24 Pic AAK00464 Mucinase 1,371 34 42 37 22 SepA CAC05786 Invasion 1,364 33 39 35 23 EpeA AY258503 Mucinase 1,359 32 48 33 22 App CAC1467 Adhesin 1,449 28 20 31 20 Hap AAX87307 Adhesin 1,436 28 16 31 20 - TABLE 4.
Prevalence of ral and rpeA among different strains of REPEC
Strain Serogroup Presence (+) or absence (−) of: eaea rpeA ral REPEC 83/39 O15:H− + + + REPEC RDEC-1 O15:H− + + − REPEC 83/146 O153:H7 + + + REPEC 84/110/1 O103:H2 + + − REPEC B10 O103:H2 + − − REPEC 82/123 O109:H2 + + + REPEC 82/90 O132:H2 + + + REPEC 82/260 O20:H7 + − − REPEC 82/183 O128:H2 + − − REPEC E22 O103:H2 + + + ↵ a Marker for the LEE pathogenicity island.
