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and PV Desmond have no conflicts of interest to declare. P De Cruz has received travel grant support from Abbott and Schering-Plough. MA Kamm has acted as an advisor to Abbott and Janssen, has received research support from Abbott, and has acted as a speaker at symposiums sponsored by Abbott and Janssen. A Hamilton has received an educational grant from Abbott. D Liew has served on advisory boards and received research grants from Abbott. IC Lawrance has been on an advisory board for Abbott and Janssen, a speaker for Abbott and Janssen, and has held research and travel grants from Abbott and Janssen. JM Andrews has been an advisory board member for both Janssen and Abbott, spoken for both Abbott and Janssen, received research funds from both Abbott and Janssen, and received travel grants from both Abbott and Janssen. PA Bampton has been on advisory boards for Janssen and Abbott, has received research funding from Abbott, and travel sponsorship from both Abbott and Janssen. PR Gibson has received consulting fees from Abbott, Janssen, and Schering-Plough; research support from Abbott; and payments for lectures from Abbott and Janssen. FA Macrae has been on an advisory board to Janssen, has received travel grants from Abbott, and has received clinical research support from Janssen, Abbott and MSD. W Selby has been on an advisory board for Abbott. SJ Bell has received travel assistance from Abbott. SJ Brown has received travel support and speaker fees from both Abbott and Janssen. WR Connell has been on advisory board for Janssen and a speaker for Abbott and Janssen. AS Day has been an M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT advisor to Janssen. RB Gearry has been on an advisory board for Abbott and Janssen, a speaker for Abbott and Janssen, and held research, educational and travel grants from Abbott and Janssen.

The function of the rorf2 gene located on the locus of enterocyte effacement (LEE) pathogenicity island of enteropathogenic Escherichia coli (EPEC) has not been described. We report that rorf2 encodes a novel protein, named EspG, which is secreted by the type III secretory system and which is translocated into host epithelial cells. EspG is homologous with Shigella flexneri protein VirA, and the cloned espG (rorf2) gene can rescue invasion in a Shigella virA mutant, indicating that these proteins are functionally equivalent in Shigella. An EPEC espG mutant had no apparent defects in in vitro assays of virulence phenotypes, but a rabbit diarrheagenic E. coli strain carrying a mutant espG showed diminished intestinal colonization and yet diarrheal attack rates similar to those of the wild type. A second EspG homolog, Orf3, is encoded on the EspC pathogenicity islet. The cloned orf3 gene could also rescue invasion in a Shigella virA mutant, but an EPEC espG orf3 double mutant was not diminished in any tested in vitro assays for EPEC virulence factors. Our results indicate that EspG plays an accessory but as yet undefined role in EPEC virulence that may involve intestinal colonization.

SummaryClostridium septicum is the causative agent of spontaneous gas gangrene or atraumatic myonecrosis, a sudden and frequently fatal infection that is increasingly associated with malignancy of the colon. Little is known about the disease process although the focus of virulence studies has been the a a a a -toxin, a pore-forming cytolysin that is encoded by the csa gene and secreted as an inactive protoxin. Until now a lack of techniques for the genetic manipulation of C. septicum has hindered the use of molecular approaches to understand pathogenesis. By introducing plasmids by conjugation from Escherichia coli , we have developed methods for the genetic manipulation of C. septicum and constructed a chromosomal csa mutant by allelic exchange. Virulence testing of an isogenic series of strains consisting of the wild type, the csa mutant, and a csa mutant complemented with the wild-type csa gene revealed that the development of fulminant myonecrosis in mice was dependent on the ability to produce a functional haemolytic a a a a -toxin. Furthermore, the inhibition of leukocyte influx into the lesion, which is very typical of clostridial myonecrosis, was also dependent on the ability to produce a a a atoxin. This study represents the first definitive identification of a virulence factor in this organism and opens the way for further studies that will delineate the role of other putative virulence factors in this significant pathogen.

Genetic manipulation has revolutionized research in the Apicomplexan parasite Plasmodium falciparum, the most important causative agent of malaria. However, to date no techniques have been established that allow modifications that are deleterious to bloodstage growth, such as the disruption of essential genes or the expression of dominant-negative transgenes. The recent establishment of a screen for functional transactivators in the related parasite Toxoplasma gondii prompted us to identify transactivators in T. gondii and to examine their functionality in P. falciparum. Tetracycline-responsive minimal promoters were generated based on the characterized P. falciparum calmodulin promoter and used to assess transactivators in P. falciparum. We demonstrate that artificial tetracycline-regulated transactivators isolated in T. gondii are also functional in P. falciparum. By using the tetracycline analogue anhydrotetracycline, efficient, stage-specific gene regulation was achieved in P. falciparum. This regulatable expression technology has clear potential for the study of essential gene function in P. falciparum blood stages. On the other hand, the identified transactivators are not functional in mammalian cells, consistent with the fundamental differences in the mechanism of gene regulation between Apicomplexan parasites and their human hosts.

Summary In this study, we detected multiple var gene transcripts within single, mature trophozoite‐infected red blood cells (iRBCs) bound to chondroitin sulphate A (CSA). Several of the var detected had previously been demonstrated to encode Plasmodium falciparum erythrocyte membrane protein‐1 (PfEMP‐1) variants with domains that mediated iRBC adhesion to receptors other than CSA. Parasites expressing the CSA‐adherent phenotype transcribed far more of one var than of all others, but this gene was different from the two other var previously purported to encode adhesion to CSA. Previous work suggesting that only single var are transcribed by mature trophozoites needs re‐examination in the light of these data from single, infected cells.

PfCDPK1 [Plasmodium falciparum CDPK1 (calcium-dependent protein kinase 1)] is highly expressed in parasite asexual blood and mosquito stages. Its role is still poorly understood, but unsuccessful gene knockout attempts suggest that it is essential for parasite replication and/or RBC (red blood cell) invasion. In the present study, by tagging endogenous CDPK1 with GFP (green fluorescent protein), we demonstrate that CDPK1 localizes to the parasite plasma membrane of replicating and invasive forms as well as very young intracellular parasites and does not appear to be exported into RBCs. Although a knockdown of endogenous CDPK1 was achieved using a destabilization domain, parasites tolerated reduced expression without displaying a phenotype. Because of this, the PfCDPK1 auto-inhibitory J (junction) domain was explored as a means of achieving inducible and specific inhibition. Under in vitro conditions, a fusion protein comprising a J-GFP fusion specifically bound to PfCDPK1 and inhibited its activity. This fusion protein was conditionally expressed in P. falciparum asexual blood stages under the regulation of a DD (destabilization domain) (J-GFP-DD). We demonstrate that J-GFP-DD binds to CDPK1 and that this results in the arrest of parasite development late in the cell cycle during early schizogony. These data point to an early schizont function for PfCDPK1 and demonstrate that conditionally expressing auto-inhibitory regions can be an effective way to address the function of Plasmodium enzymes.

SUMMARY BackgroundCrohn's disease recurs in the majority of patients after intestinal resection.