Abstract
Uropathogenic Escherichia coli (UPEC) are the etiologic agent of more than 80% of
uncomplicated urinary tract infections (UTIs) in humans. Pyelonephritis-associated
pili (Pap) are fimbrial adhesins that facilitate binding of UPEC to Gala-(l,4)-Gal/i
moieties contained in membrane glycolipids on human uroepithelial cells and are
associated with acute kidney infection (pyelonephritis). Pap expression is phase
variable and the frequency with which phase transition occurs determines the
proportion of P-fimbriate bacteria in the population. In this study, pap 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 pap operons
measured in E. coli K-12. In this relevant regulatory context, phase variation
frequencies of homologous pap operons were found to be differentially affected by
culture conditions, indicating a hierarchy of expression depending on environmental
signals. Cross-talk between pap operons was also found to be dependent on culture
conditions. The molecular mechanism leading to different phase variation
frequencies between homologous pap operons was investigated by sequencing 82
pap regulatory regions and their regulators (papl and papB) from 54 UPEC isolates
of different clinical origin (asymptomatic vs. symptomatic UTI). One variable region
identified was a high affinity binding site for the pap autoregulatory protein PapB.
The site contained a variable number of 9 bp repeats with (T/A)3 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 papl genes, encoding for the pap transcriptional activator Papl,
and were shown to be under positive selection (dN/dS > 1) for functionally adaptive
amino acid replacements. Certain Papl variants correlated with symptomatic disease
and differed in their ability to activate pap operons. This study provided different
lines of evidence supporting the hypothesis that UPEC isolates have evolved
mechanisms to regulate the phase variation frequencies of homologous fimbrial
operons, potentially to achieve their differential activation and sequential expression.
The ability of UPEC to coordinate expression of multiple surface factors is critical
for the successful colonisation of the varied complex micro-environments
encountered in the human urinary tract.
