Cytotoxic necrotizing factor type 1 (CNF1) is one of the virulence factors produced by uropathogenic Escherichia coli (UPEC). How this toxin is translocated from the bacterial cytoplasm to the surrounding environment is not well understood. Our data suggest that CNF1 may be regarded as a secreted protein, since it could be detected in culture supernatants. Furthermore, we found that CNF1 was tightly associated to outer membrane vesicles, suggesting that such vesicles play a role in the secretion of this protein. Interestingly, vesicle samples containing CNF1 could exert the effects known for this protein on HeLa cell cultures, showing that CNF1 is transported by vesicles in its active form. Taken together, our results strongly suggest that outer membrane vesicles could be a means for the bacteria to deliver CNF1 to the environment and to the infected tissue. In addition, our results indicate that the histone-like nucleoid structuring protein H-NS has a role in the downregulation of CNF1 production and that it affects the outer membrane vesicle release in UPEC strain J96.Uropathogenic Escherichia coli (UPEC) strains are responsible for at least 80% of human urinary tract infections (UTIs), which include urethritis, cystitis, and pyelonephritis (21). UTI is among the most common bacterial infections in humans and has been shown to be an independent risk factor for both bladder cancer and renal cell carcinoma (30). To achieve pathogenicity, the UPEC strains produce many virulence factors such as adhesins (P and type 1 pili), hemolysin, aerobactin siderophore system, a capsule, and cytotoxic necrotizing factor type 1 (CNF1). CNF1 belongs to a group of bacterial necrotic substances which include the dermonecrotic toxin of Bordetella spp. (14), the virulence plasmid-encoded CNF2 of E. coli (14,29), and the recently described CNFy from Yersinia pseudotuberculosis (24). Unlike CNFy, whose mechanism of action is specific to RhoA (13), CNF1 affects three proteins of the Rho family of the small GTPases: RhoA, Rac1, and Cdc42. CNF1 deamidates Gln63 of RhoA (or the corresponding Gln61 of Rac1 and Cdc42), leading to the loss of both intrinsic and GAP-stimulated GTPase activities but without affecting the GTP-binding activity (14). This modification renders these GTPases constitutively active, which commonly results in formation of cellular actin stress fibers, filopodia and lamellipodia, with an overall effect of blocking the cytokinesis, leading to giant multinucleated cells (7,23,25). CNF1 is a 115-kDa A-B toxin, in which a catalytic A domain is located in the C-terminal region of about 300 amino acids and where the cell binding B-domain is in the N-terminal region (amino acids 53 to 190) (6,22). No typical signal peptide is found in the CNF1 sequence, and the secretion pathway of this toxin remains unclear. In this work we show that CNF1 is secreted and that it is associated with membrane vesicles. Furthermore, we present evidence that CNF1 is down regulated by H-NS (histone-like nucleoid structuring protein). MATERIALS AN...

The essential oil and several pure sulfur compounds isolated from Scorodophloeus zenkeri were tested for their antibacterial and antifungal activity using a paper disc method, the poisoned food technique, a microatmosphere method and the measurement of cellular ATP content. The essential oil completely inhibited the growth of all fungi tested including yeasts, with the exception of Aspergillus flavus, and was active against the Gram-positive bacteria studied, but not the Gram-negative organisms. 2,4,5,7-Tetrathiaoctane, 2,4,5,6,8-pentathianonane, 2,3,4,6,8-pentathianonane, 2,3,5,6,8,10-hexathiaundecane, 2,3,5-trithiahexane 5-oxide, 2,4,5,7-tetrathiaoctane 2-oxide, 2,3,5,7-tetrathiaoctane 3,3-dioxide and 2,3,5-trithiahexane 3,3-dioxide differed in their effects on the strains studied with respect to both growth and synthesis of cellular ATP. 2,3,5-Trithiahexane, 2,3,4,6-tetrathiaheptane, methyl methanethiosulfonate and bis-methyl-sulfonylmethane exhibited no antimicrobial activity.

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2,3,5-trithiahexane, 2,3,4,6-tetrathiaheptane, 2,4,5,7-tetrathiaoctane, two pentathianonanes, 2,4,5,7,9-pentathiadecane and two hexathiaundecanes were isolated from the essential oil and extracts from the bark of Scorodophloeus zenkeri Harms. Four other thioalkanes were found in small amounts in the essential oil.