Pch Genes Control Biofilm and Cell Adhesion in a Clinical Serotype O157:H7 Isolate

Abstract

In a previous study, induction of the Escherichia coli serotype O157:H7 SOS response decreased csgD expression in the clinical isolate PA20 at 30°C but strongly induced genes in the horizontally transferred-DNA regions (HTR), including many known virulence regulators. To determine the role of HTR regulators in the control of csgD and curli, specific regulators were plasmid-expressed in the wild-type and mutant strains of PA20 and its biofilm-forming derivative, 20R2R. At 30°C, plasmid over-expression of the O157:H7 group 3 perC homolog, pchE, strongly repressed PA20 csgD transcription (>7-fold) while the group 1 homologs, pchA and pchB, resulted in smaller reductions (<2.5-fold). However, SOS induction decreased rather than increased pchE expression (>6-fold) making group 1 pch, which are enhanced by the SOS response, the likely SOS-induced csgD repressors. Plasmid-based pchE over-expression also reduced 20R2R biofilm formation (>6-fold) and the curli-dependent, Congo red affinity of both PA20 and 20R2R. However, to properly appreciate the regulatory direction, expression patterns, and environmental consequences of these and other CsgD-controlled functions, a better understanding of natural pchE regulation will be required. The effects of HTR regulators on PA20 and 20R2R adhesion to HEp-2 cell at host temperature were also studied. Under conditions where prophage genes were not induced, curli, rather than espA, contributed to host cell adhesion in strain 20R2R. High levels of pchE expression in trans reduced curli-dependent cell adherence (>2-fold) to both 20R2R and the clinical isolate PA20, providing a host-adapting adhesion control mechanism. Expression of pchE was also repressed by induction of the SOS response at 37°C, providing a mechanism by which curli expression might complement EspA-dependent intimate adhesion initiated by the group1 pch homologs. This study has increased our understanding of the O157 pch genes at both host and environment temperatures, identifying pchE as a strong regulator of csgD and CsgD-dependent properties.

PchE Regulation of Escherichia coli O157:H7 Flagella, Controlling the Transition to Host Cell Attachment

“…The activation of LEE and intimate cell attachment are well described properties of group 1 perC homologues, but the group 2 ( pchD ) and 3 ( pchE ) genes have been less well characterized [ 13 , 14 , 44 ]. This and previous studies have indicated that pchE functions in opposition to class 1 genes to manage cellular adhesion, likely in a temporal manner where pchE balances general cell adherence and motility prior to LEE activation and the initiation of stronger intimate contacts [ 38 , 42 ]. Earlier, we showed that PA20 expresses a low steady-state level of pchE that is reduced when prophage elements are induced [ 42 ].…”

“…Our earlier studies showed that the expression of pchE reduced PA20 attachment to HEp-2 cells by repressing curli under conditions where the SOS response was not stimulated [ 38 ]. The findings also indicated that pchE controlled additional adhesins involved in cell attachment.…”

“…PchE controls curli in part by a strong repressive effect on the curli master regulator, csgD [ 38 ]. We found here that PchE also affects the expression of the flagellar master regulator genes flhDC , probably mediating some, if not all, of the flagellar changes.…”

“…Strains PA20Δ csgD and PA20Δ pchE were constructed by Red/ET recombination replacing the open-reading-frame with a neomycin cassette. The cloning of pchE into inducible plasmid pSE380 (PA20 pSE380:: pchE ) has been described in a different study [ 38 ]. Strains of PA20 expressing pchE from pSE380 were grown without IPTG induction relying on copy-number and leaky expression from the trc promoter for protein production.…”

“…However, csgD is also expressed at 37 °C, by a mechanism that has yet to be clearly defined, and it can play a role in attachment to cultured tissue cells and animal GI tissues. A recent study showed that csgD and curli ( csgA ) were strongly downregulated by plasmid over-expression of pchE and that a steady natural level of pchE in O157:H7 was repressed by SOS induction following treatment with sulfamethoxazole/trimethoprim (SMX-TM) at 540/108 μg/L [ 38 ]. In that study, pchE repressed HEp-2 cell adhesion to lower levels than those observed following curli subunit deletion indicating that there may be additional adhesins in the pchE regulon.…”