The Cytotoxic Necrotizing Factors (CNFs)-A Family of Rho GTPase-Activating Bacterial Exotoxins

Abstract

The cytotoxic necrotizing factors (CNFs) are a family of Rho GTPase-activating single-chain exotoxins that are produced by several Gram-negative pathogenic bacteria. Due to the pleiotropic activities of the targeted Rho GTPases, the CNFs trigger multiple signaling pathways and host cell processes with diverse functional consequences. They influence cytokinesis, tissue integrity, cell barriers, and cell death, as well as the induction of inflammatory and immune cell responses. This has an enormous influence on host-pathogen interactions and the severity of the infection. The present review provides a comprehensive insight into our current knowledge of the modular structure, cell entry mechanisms, and the mode of action of this class of toxins, and describes their influence on the cell, tissue/organ, and systems levels. In addition to their toxic functions, possibilities for their use as drug delivery tool and for therapeutic applications against important illnesses, including nervous system diseases and cancer, have also been identified and are discussed.

Keywords: CNF; E. coli; Rho-GTPases; Yersinia; cytotoxic necrotizing factors; inflammation.

Figure 1

Domain structure and organization of CNF_Y and its derivatives. (A) Sequence comparison of the cnfY gene from the Y. pseudotuberculosis strains YPIII, 713425, and IP2777, and Y. pestis strain CO92. The C-terminal and internal deletions of the cnfY derivates are indicated. The amino acid number of the domain ends according to the amino acid sequence are given. D1-D5 illustrate the individual domains. (B) Representation of CNF_Y crystal structure colored according to the domain boundaries shown in (A). Domain D1 (dark purple) includes the p37LRP/67LR receptor-binding motif with predicted membrane-inserting a-helices (light purple); linker domain between D4–5 contains the second binding site. The cleavage site, which is used to separate D4–5 from the full-length CNF_Y protein upon the release into the cytoplasm is indicated. (C) Representation of the two conformations of the D4–5, in the full-length and the cleaved state. The active deamidase site exposed in the cleaved derivative is indicated by an arrow.

Figure 2

Uptake and intracellular trafficking of CNFs. The CNFs are secreted directly and/or bound on outer membrane vesicles. (1) They interact with two different cell receptors with the N-terminal and C-terminal domains, and (2) internalize into host cells by endocytosis and are found in the early endosomes (3). Acidification of the late endosome induces a conformational change (4) and proteolytic processing (5), releasing the catalytic fragment D4–5 into the cytosol (6). The D4–5 protein fragment deamidates Rho GTPases at the cell membrane (7), which triggers multiple cell responses, including ubiquitinylation and proteolytic degradation (8).

Figure 3

CNF1-induced signaling pathways and host cell processes. Overview of the CNF1-triggered signaling pathways through the activation of the small Rho GTPases RhoA, Rac1, and Cdc42, leading to (I) actin cytoskeleton rearrangements and modulation of cell barriers, (II) interferences with cytokinesis and cell death programs, and (III) modulation of proinflammatory cytokines.