LRP mediated transcriptional activation of PapR.

<p>(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), UTI89Δ<i>lrp</i>/pNDM220 (lane 3) and UTI89Δ<i>hfq</i>/pNDM220 (lane 5) with the respective complemented strains UTI89Δ<i>lrp</i>/pSKlrp (lane 2) and UTI89Δ<i>hfq</i>/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 <i>papR</i> transcription.</p

Hfq impact on bladder cell infection.

<p>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. UTI89Δ<i>hfq</i> showed a statistically significant reduction in adhesion as well as invasion (*<i>p-value</i> <0.05).</p

Model illustrating PapR-mediated modulation of P-fimbriae phase variation.

<p>The <i>pap</i> 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 <i>papI</i> 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.</p

Characterization of PapR function.

<p>(A) Heme- and yeast cell agglutination assays performed with UTI89/pNDM220, UTI89Δ<i>papR</i>/pNDM220 and UTI89Δ<i>papR</i>/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, UTI89Δ<i>papR</i>/pNDM220, UTI89Δ<i>papR</i>/pSK1 and UTI89Δ<i>hfq</i>. 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 (*<i>p-value</i> <0.05). (C) Flow cytometry of UTI89/pNDM220, UTI89Δ<i>papR</i>/pNDM220 and UTI89Δ<i>papR</i>/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 (*<i>p-value</i> <0.05).</p

Novel sRNAs detected in UTI89.

<p>(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.</p

DamX Controls Reversible Cell Morphology Switching in Uropathogenic Escherichia coli

The ability to change cell morphology is an advantageous characteristic adopted by multiple pathogenic bacteria in order to evade host immune detection and assault during infection. Uropathogenic Escherichia coli (UPEC) exhibits such cellular dynamics and has been shown to transition through a series of distinct morphological phenotypes during a urinary tract infection. Here, we report the first systematic spatio-temporal gene expression analysis of the UPEC transition through these phenotypes by using a flow chamber-based in vitro infection model that simulates conditions in the bladder. This analysis revealed a novel association between the cell division gene damX and reversible UPEC filamentation. We demonstrate a lack of reversible bacterial filamentation in a damX deletion mutant in vitro and absence of a filamentous response by this mutant in a murine model of cystitis. While deletion of damX abrogated UPEC filamentation and secondary surface colonization in tissue culture and in mouse infections, transient overexpression of damX resulted in reversible UPEC filamentation. In this study, we identify a hitherto-unknown damX-mediated mechanism underlying UPEC morphotypical switching. Murine infection studies showed that DamX is essential for establishment of a robust urinary tract infection, thus emphasizing its role as a mediator of virulence. Our study demonstrates the value of an in vitro methodology, in which uroepithelium infection is closely simulated, when undertaking targeted investigations that are challenging to perform in animal infection models