Expression of cnf1 by Escherichia coli J96 involves a large upstream DNA region including the hlyCABD operon, and is regulated by the RfaH protein

Abstract

Summary Examination of 55 clinical isolates of uropathogenicEscherichia coli producing the CNF1 toxin demonstrated that the cnf1 gene is systematically associated with a hly operon via a highly conserved hlyDcnf1 intergenic region ( igs , 943 bp) as shown in the J96 UPEC strain. We examined if this association could reflect a co-regulation of the production of these toxins. Translation of cnf1 from an immediately upstream promoter has been shown to be controlled by means of an anti-Shine-Dalgarno sequence present in the cnf1 coding sequence [fold-back inhibition ( cnf1 fbi )]. The cnf1 fbi was not regulated by elements present in the igs . An RNA covering the full hlyD sequence, the igs and extending on the cnf1 gene, was then detected in the J96 strain. This RNA could be part of a HlyCABD mRNA. Transcription of the haemolysin operon requires RfaH antitermination activity. Inactivation of rfaH in J96 resulted in a 100-fold reduction of the CNF1 content of bacteria. The production of CNF1 from a plasmidic igscnf1 DNA was not sensitive to RfaH, indicating that this factor acted on cnf1 transcription via the hly promoter. This way the cnf1 fbi mechanism might be overcome by transcription of cnf1 from the haemolysin promoter and antitermination by RfaH. This constitutes a novel system of bacterial virulence factors co-regulation.

Oral Immunization with an rfaH Mutant Elicits Protection against Salmonellosis in Mice

“…Later RfaH was shown to be essential for the expression of other cell components encoded on long operons in E. coli. RfaH-affected operons include those encoding the F plasmid (26), different capsules (4,23,30), and hemin uptake receptor (18), as well as the toxins alpha-hemolysin (3,16) and cytotoxic necrotizing factor 1 (CNF-1) (15). A global regulatory role of RfaH in the virulence of uropathogenic E. coli has recently been shown (19).…”

Hemolysin of Uropathogenic Escherichia coli Evokes Extensive Shedding of the Uroepithelium and Hemorrhage in Bladder Tissue within the First 24 Hours after Intraurethral Inoculation of Mice

“…As mentioned above, CP9 carries two hly genes. The hlyA 1 gene is located in an operon upstream of cnf 1 (unpublished observation), as it is in other UPEC strains, such as J96 (6,33,37,62). In this investigation, we introduced a nonpolar mutation in hlyA 1 into the isogenic CP9cnf 1 mutant strain by using the lambda red recombination technique (12), with the modifications described below.…”

“…Moreover, Real and coworkers reported that CNF1/Hly-expressing UPEC strains are more often isolated from humans with hemorrhagic UTIs who also have higher urinary levels of proinflammatory cytokines, such as interleukin-8 and monocyte chemoattractant protein 1, than UPEC strains that do not make these toxins (51). Landraud et al provided a genetic explanation for the apparent linkage between CNF1 and Hly synthesis by UPEC; they demonstrated that a cnf 1 gene and an hly operon in the prototypic UPEC strain J96 not only are cotranscribed but also are coregulated in vitro (37).…”

Novel Three-Dimensional Organoid Model for Evaluation of the Interaction of Uropathogenic Escherichia coli with Terminally Differentiated Human Urothelial Cells

“…CP9 carries two hemolysin genes (10); one hlyA copy is located within an operon immediately upstream of cnf 1 (unpublished observation), as is seen in other uropathogenic E. coli strains, such as J96 (04/H5/K Ϫ ) (1,10,12,24). In this study, we introduced a nonpolar mutation in hlyA upstream of cnf 1 by the Lambda Red recombination technique (3) with modifications as noted below.…”