| Summary: | Pyelonephritis-associated pili (Pap) are fimbrial adhesions that facilitate binding of UPEC to Gal(α 1-4)Galβ moieties contained in membrane glycolipids on human uroeipthelial cells and are associated with acute kidney infection (pyelonephritis). In this study, <i>pap</i> phase transition frequencies were measured in clinical isolates for the first time and were shown to be markedly higher than the frequencies displayed by the same <i>pap</i> operons measured in <i>E. coli</i> K-12. In this relevant regulatory context, phase variation frequencies of homologous <i>pap</i> operons were found to be differentially affected by culture conditions, indicating a hierarchy of expression depending on environmental signals. Cross-talk between <i>pap</i> operons was also found to be dependent on culture conditions. The molecular mechanism of different phase variation frequencies between homologous <i>pap</i> operons was investigated by sequencing 82 <i>pap</i> regulatory regions and their regulators (<i>papI </i>and <i>papB</i>) from 54 UPEC isolates of different clinical origin (asymptomatic <i>vs.</i> symptomatic UTI). The most variable region identified was a high affinity binding site for the <i>pap</i> autoregulatory protein PapB. The site contained a variable number of 9 bp repeats with (T/A)<sub>3</sub> sequences, which affected PapB binding and the frequency of off-to-on phase transition., under particular environmental conditions. Sequence diversification via point mutation was also observed among <i>papI</i> genes, encoding for the <i>pap</i> transitional activator PapI, and were shown to be under positive selection (Dn/Ds > 1) for functionally adaptive amino acid replacements. Certain PapI variants correlated with symptomatic disease and differed in their ability to activate <i>pap</i> operons. The ability of UPEC to co-ordinate expression of multiple surface factors is critical for the successful colonisation of the many complex micro-environments encountered in the human urinary tract.
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