Fig 1.
Hfq impact on bladder cell infection.
PD07i bladder cells cultured in 24-well plates were infected with UPEC strains cultured overnight at 37°C in LB medium. Mean CFU counts of triplicates from three independent experiments were plotted and standard deviation calculated. UTI89Δhfq showed a statistically significant reduction in adhesion as well as invasion (*p-value <0.05).
Fig 2.
Differential sRNA expression during infection.
Graphical representation of percentage coverage of normalized sequence reads from culture reference and infection cDNA libraries corresponding to known sRNAs.
Fig 3.
Novel sRNAs detected in UTI89.
(A) Northern blots showing transcript levels for PapR and C271. Lanes 1 and 2 represent RNA from culture reference and infection respectively. (B) Graphical output of normalized sequence reads mapping to PapR and C271 in culture reference and infection visualized using Integrated Genome Viewer (Broad Institute). The table below includes normalized total read counts within the PapR and C271 probes.
Fig 4.
LRP mediated transcriptional activation of PapR.
(A) PapR transcriptional start site was mapped using primer extension reaction loaded in the lane marked 1 alongside Sanger sequencing reactions for the four nucleotides represented in lanes GCAT. (B) Northern blotting was used to detect PapR levels in UTI89/pNDM220 (lane 1), UTI89Δlrp/pNDM220 (lane 3) and UTI89Δhfq/pNDM220 (lane 5) with the respective complemented strains UTI89Δlrp/pSKlrp (lane 2) and UTI89Δhfq/pJMJ220 (lane 4), all cultured in LB medium. 5S RNA was used as the internal loading control. (C) Illustration of the genomic context of PapR in the UTI89 genome, drawn to scale. LRP was found to positively regulate papR transcription.
Fig 5.
Characterization of PapR function.
(A) Heme- and yeast cell agglutination assays performed with UTI89/pNDM220, UTI89ΔpapR/pNDM220 and UTI89ΔpapR/pSK1 with and without the addition of α-D-mannose. Scale bars set at 50 μm. (B) PD07i bladder cells and IMCD3 kidney collecting duct cells cultured in 24-well plates were infected with UTI89/pNDM220, UTI89ΔpapR/pNDM220, UTI89ΔpapR/pSK1 and UTI89Δhfq. Strains were either left untreated (-) or treated (+) with 3% α-D-mannose and used for infection. Bacterial adhesion was assessed by calculating mean CFU counts from three independent experiments. Statistical significance was calculated using Students t-test (*p-value <0.05). (C) Flow cytometry of UTI89/pNDM220, UTI89ΔpapR/pNDM220 and UTI89ΔpapR/pSK1 grown overnight in LB medium and immunolabelled with α-PapA and α-Fim antibodies was used to detect the extent of P- and type-1 fimbriated cells respectively. Mean fluorescence from three independent experiments was plotted along with the standard deviations. Statistical significance was calculated using Students t-test (*p-value <0.05).
Fig 6.
PapR mediated papI repression.
(A) β-galactosidase assays were performed with DL1504 strains with and without papI. Induction of PapR using 1mM IPTG resulted in a 50% reduction in β-galactosidase activity in DL1504 (*p-value <0.05) but not DL1504ΔpapI. Mean values from three independent experiments were plotted and standard deviations calculated. (B) PapR was induced with 1 mM IPTG in DL2121 strains and mean values of β-galactosidase activity measured in Miller units were plotted. (C) Validation of papI as a PapR target was performed using Top10/pSKpapI/pNDM220 (lane 1), Top10/pSKpapI/pSK1 (lanes 2–3) and Top10/pSKpapI/pSK1* (lanes 4–5). Strains were grown with (lanes 1, 2 and 4) and without (lanes 3 and 5) the presence of 1 mM IPTG to induce PapR from pSK1 and PapR* from pSK1*. Northern blot detection of PapR, PapR* and papI::gfp levels was carried out in parallel with Western blot detection of PapI::GFP. GroEL and 5S were used as internal controls in the Western and Northern blots respectively.
Fig 7.
Model illustrating PapR-mediated modulation of P-fimbriae phase variation.
The pap gene cluster encodes two regulatory proteins, PapI and PapB, that work in concert with other global regulators such as LRP, H-NS and Dam methylase to control P-fimbrial phase variation between the OFF and ON states. LRP mediated transcriptional activation of PapR sRNA results in the degradation of papI mRNA. An absence of functional PapI results in a failure to switch from an OFF to an ON phase, and a failure therein to express P-fimbriae on the surface.