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Infection and Immunity, October 2003, p. 5900-5909, Vol. 71, No. 10
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.10.5900-5909.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Heterogeneous Surface Expression of EspA Translocon Filaments by Escherichia coli O157:H7 Is Controlled at the Posttranscriptional Level

Andrew J. Roe,1 Helen Yull,1 Stuart W. Naylor,1 Martin J. Woodward,2 David G. E. Smith,1 and David L. Gally1*

Zoonotic and Animal Pathogens Research Laboratory, Medical Microbiology, University of Edinburgh, Edinburgh EH8 9AG,1 Veterinary Laboratory Agency, Addlestone, Surrey KT15 3NB, United Kingdom2

Received 17 March 2003/ Returned for modification 12 June 2003/ Accepted 24 July 2003

Type III secretion systems of enteric bacteria enable translocation of effector proteins into host cells. Secreted proteins of verotoxigenic Escherichia coli O157 strains include components of a translocation apparatus, EspA, -B, and -D, as well as "effectors" such as the translocated intimin receptor (Tir) and the mitochondrion-associated protein (Map). This research has investigated the regulation of LEE4 translocon proteins, in particular EspA. EspA filaments could not be detected on the bacterial cell surface when E. coli O157:H7 was cultured in M9 minimal medium but were expressed from only a proportion of the bacterial population when cultured in minimal essential medium modified with 25 mM HEPES. The highest proportions of EspA-filamented bacteria were detected in late exponential phase, after which filaments were lost rapidly from the bacterial cell surface. Our previous research had shown that human and bovine E. coli O157:H7 strains exhibit marked differences in EspD secretion levels. Here it is demonstrated that the proportion of the bacterial population expressing EspA filaments was associated with the level of EspD secretion. The ability of individual bacteria to express EspA filaments was not controlled at the level of LEE1-4 operon transcription, as demonstrated by using both ß-galactosidase and green fluorescent protein (GFP) promoter fusions. All bacteria, whether expressing EspA filaments or not, showed equivalent levels of GFP expression when LEE1-4 translational fusions were used. Despite this, the LEE4-espADB mRNA was more abundant from populations with a high proportion of nonsecreting bacteria (low secretors) than from populations with a high proportion of secreting and therefore filamented bacteria (high secretors). This research demonstrates that while specific environmental conditions are required to induce LEE1-4 expression, a further checkpoint exists before EspA filaments are produced on the bacterial surface and secretion of effector proteins occurs. This checkpoint in E. coli O157:H7 translocon expression is controlled by a posttranscriptional mechanism acting on LEE4-espADB mRNA. The heterogeneity in EspA filamentation could arise from phase-variable expression of regulators that control this posttranscriptional mechanism.

* Corresponding author. Mailing address: Zoonotic and Animal Pathogens Research Laboratory, Medical Microbiology, Teviot Place, University of Edinburgh, Edinburgh EH8 9AG, United Kingdom. Phone: 00 44 131 6511342. Fax: 00 44 131 6506531. E-mail: d.gally{at}ed.ac.uk.

Editor: V. J. DiRita

Infection and Immunity, October 2003, p. 5900-5909, Vol. 71, No. 10
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.10.5900-5909.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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