Luce Landraud

Activation and Proteasomal Degradation of Rho GTPases by Cytotoxic Necrotizing Factor-1 Elicit a Controlled Inflammatory Response

The CNF1 toxin is produced by uropathogenic and meningitis-causing Escherichia coli. CNF1 penetrates autonomously into cells and confers phagocytic properties to epithelial and endothelial cells. CNF1 acts at the molecular level by constitutively activating Rho GTPases attenuated by their cellular ubiquitin-mediated proteasomal degradation. Here we report the relationship between the ubiquitin-mediated proteasomal degradation of activated Rho and the endothelial cell response to the toxin. The type of cellular response to CNF1 intoxication, first screened by DNA microarray analysis, revealed the launching of a program oriented toward an inflammatory response. Parallel to Rho protein activation by CNF1, we also established the kinetics of production of monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), IL-6, monocyte inflammatory protein-3α (MIP-3α) and E-selectin. Both the mutation of the catalytic domain of the toxin (CNF1-C866S) and the inhibition of Rho proteins abrogate the CNF1-induced production of the immunomodulators MIP-3α, MCP-1, and IL-8. These immunomodulators are also produced upon activation of Cdc42 and Rac preferentially. Our results indicate that, in addition to pathogen molecular pattern recognition by host-receptors, a direct activation of Rho proteins by the CNF1 virulence factor efficiently triggers a cellular reaction of host alert. Consistently, we assume that the CNF1-induced ubiquitin-mediated proteasomal degradation of activated Rho proteins may limit the amplitude of the host cell immune responses.

Frequency of Escherichia coli strains producing the cytotoxic necrotizing factor (CNF1) in nosocomial urinary tract infections.

The presence of cytotoxic necrotizing factor 1 (CNF1), together with various associated virulence factors (alpha‐haemolysin, P‐, S‐ and A‐fimbriae), was screened in 175 uropathogenic Escherichia coli strains isolated from hospitalized adult patients. The cnf1 gene was detected in 30% of the selected strains independently of the severity of the clinical urinary infection. A significant association between CNF1, haemolytic activity and the products of the pap/sfa genes was found. However, CNF1 appeared not to play a major role in nosocomial E. coli urinary tract infections..

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

Examination of 55 clinical isolates of uropathogenic Escherichia coli producing the CNF1 toxin demonstrated that the cnf1 gene is systematically associated with a hly operon via a highly conserved hlyD‐cnf1 intergenic region (igs, 943u2003bp) 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.

E. coli CNF1 toxin: a two-in-one system for host-cell invasion

The cytotoxic necrotizing factor-1 (CNF1), a bacterial toxin of uropathogenic bacteria (UPEC), hijacks cellular Rho proteins of the Ras GTPase super-family. Recently, we have made three important findings. First, we have established that, following Rho protein activation by deamidation, these cellular proteins are ubiquitylated and degraded by the proteasome. Second, the low level of activated Rho proteins which results from the dual molecular mechanism of action of CNF1 (Rho protein activation followed by their degradation), confers high invasive properties to UPECs. Finally, we have reported that ubiquitylation and degradation of Rac is lost in HEp-2 carcinoma cells, thereby suggesting a possible link between Rho protein ubiquitylation and tumor progression.

Decreased susceptibility to chlorhexidine affects a quarter of Escherichia coli isolates responsible for pneumonia in ICU patients

Béatrice La Combe, Alexandre Bleibtreu, Jonathan Messika, Romain Fernandes, Olivier Clermont, Catherine Branger, Typhaine Billard‐Pomares, Guilène Barnaud, Fatma Magdoud, Matthieu Eveillard, Achille Kouatchet, Sigismond Lasocki, Pierre Asfar, Stéphane Corvec, Karim Lakhal, Laurence Armand‐Lefevre, Michel Wolff, Jean‐François Timsit, Sandrine Bourdon, Jean Reignier, Stéphanie Martin, Vincent Fihman, Nicolas de Prost, Julien Bador, Pierre‐Emmanuel Charles, Julien Goret, Alexandre Boyer, Frederic Wallet, Emmanuelle Jaillette, Saad Nseir, Luce Landraud, Raymond Ruimy, Pierre‐Eric Danin, Jean Dellamonica, Julie Cremniter, Jean‐Pierre Frat, Françoise Jauréguy, Christophe Clec’h, Dominique Decré, Eric Maury, Didier Dreyfuss, Erick Denamur and Jean‐Damien Ricard