Two Domains of Cytotoxic Necrotizing Factor Type 1 Bind the Cellular Receptor, Laminin Receptor Precursor Protein

McNichol, Beth A.; Rasmussen, Susan B.; Carvalho, Humberto M.; Meysick, Karen C.; O'Brien, Alison D.

doi:10.1128/iai.00075-07

Cited by 29 publications

(41 citation statements)

References 34 publications

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“…The critical sequence enabling CbpA to bind to LR included the loop EPRNEEK, a region missing in the CbpA of the 2 clinical strains that failed to bind LR. While this sequence was not found in PilQ, PorA, or OmpP2, we identified the sequence NEENEEK within the domain of CNF1 toxin of E. coli that is believed to bind to LR (13).…”

Section: Discussionmentioning

confidence: 96%

“…LR on microvascular endothelial cells interacts with neurotropic viruses, including Sindbis virus (7), Dengue virus (8), adeno-associated virus (9), tick-borne encephalitis virus, and Venezuelan equine encephalitis virus (10). Additionally, LR binds the cellular prion protein PrP (11,12) and the secreted cytotoxic necrotizing factor toxin of E. coli (13,14). Thus, LR participates in diverse cell-cell and cell-pathogen interactions.…”

Section: Introductionmentioning

confidence: 99%

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Laminin receptor initiates bacterial contact with the blood brain barrier in experimental meningitis models

Mahdavi²,

et al. 2009

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A diverse array of infectious agents, including prions and certain neurotropic viruses, bind to the laminin receptor (LR), and this determines tropism to the CNS. Bacterial meningitis in childhood is almost exclusively caused by the respiratory tract pathogens Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae, but the mechanism by which they initiate contact with the vascular endothelium of the blood brain barrier (BBB) is unknown. We hypothesized that an interaction with LR might underlie their CNS tropism. Using affinity chromatography, coimmunoprecipitation, retagging, and in vivo imaging approaches, we identified 37/67-kDa LR as a common receptor for all 3 bacteria on the surface of rodent and human brain microvascular endothelial cells. Mutagenesis studies indicated that the corresponding bacterial LR-binding adhesins were pneumococcal CbpA, meningococcal PilQ and PorA, and OmpP2 of H. influenzae. The results of competitive binding experiments suggest that a common adhesin recognition site is present in the carboxyl terminus of LR. Together, these findings suggest that disruption or modulation of the interaction of bacterial adhesins with LR might engender unexpectedly broad protection against bacterial meningitis and may provide a therapeutic target for the prevention and treatment of disease.

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Section: Discussionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Laminin receptor initiates bacterial contact with the blood brain barrier in experimental meningitis models

Mahdavi²,

et al. 2009

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“…The different effects of CNF1 and CNF2 on HeLa cells may be attributed to differences in the preference of these two toxins for small GTPase substrates (34). Furthermore, McNichol et al (22) recently showed that the capacity of CNF2 to bind to HEp-2 cells was significantly reduced but that CNF2 bound to the laminin receptor precursor protein (LRP) at levels similar to those of CNF1. Together, these results provide evidence that small regions of CNF1 and CNF2 may significantly influence the phenotype of each toxin (24).…”

mentioning

confidence: 99%

“…In fact, two specific residues in the C terminus of CNF1, C866 and H881, had previously been shown to be essential for deamidation activity (7). In a third study, McNichol et al (22) proposed that there are two receptor-binding domains for CNF1, the first located in the N terminus (amino acids 135 to 164) and the second located in the C-terminal portion of CNF1 (amino acids 683 to 730). These domains were hypothesized to interact and form a three-dimensional receptor-binding domain.…”

mentioning

confidence: 99%

“…Since this region of CNF1 included one of the proposed receptor-binding domains of CNF1 (22), we also investigated whether the amino acids in the MAb NG8 epitope and the amino acids responsible for differences in CNF1 and CNF2 HEp-2 cell binding were the same. We found that when three noncontiguous amino acids from CNF1 were exchanged for the amino acids present at the same positions in CNF2, MAb NG8 recognized the chimeric CNF2 molecule with the E591D, L593F, and Q661H mutations (CNF2 E591D L593F Q661H ) and had the capacity to neutralize CNF2 intoxication of HEp-2 cells.…”

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confidence: 99%

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Cytotoxic Necrotizing Factor Type 1-Neutralizing Monoclonal Antibody NG8 Recognizes Three Amino Acids in a C-Terminal Region of the Toxin and Reduces Toxin Binding to HEp-2 Cells

Grande

Meysick²,

Rasmussen

et al. 2009

Infect Immun

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Cytotoxic necrotizing factor type 1 (CNF1) and CNF2 are toxins of pathogenic Escherichia coli that share 85% identity over 1,014 amino acids. Although both of these toxins modify GTPases, CNF1 is a more potent inducer of multinucleation in HEp-2 cells, binds more efficiently to HEp-2 cells, and, despite the conservation of amino acids (C866 and H881) required for enzymatic activity of the toxins, deamidates RhoA and Cdc42 better than CNF2. Here we exploited the differences between CNF1 and CNF2 to define the epitope on CNF1 to which the CNF1-specific neutralizing monoclonal antibody (MAb) (MAb NG8) binds and to determine the mechanism by which MAb NG8 neutralizes CNF1 activity on HEp-2 cells. For these purposes, we generated a panel of 21 site-directed mutants in which amino acids in CNF1 were exchanged for the amino acids in CNF2 between amino acids 546 and 869 and vice versa. This region of CNF1 not only is recognized by MAb NG8 but also is involved in binding of this toxin to HEp-2 cells. All the mutants retained the capacity to induce multinucleation of HEp-2 cells. However, the CNF1 double mutant with D591E and F593L mutations (CNF1 D591E F593L ) and the CNF1 H661Q single mutant displayed drastically reduced reactivity with MAb NG8. A reverse chimeric triple mutant, CNF1 E591D L593F Q661H , imparted MAb NG8 reactivity to CNF2. MAb NG8 neutralized CNF2 E591D L593F Q661H activity in a dose-dependent manner and reduced the binding of this chimeric toxin to HEp-2 cells. Taken together, these results pinpoint three amino acids in CNF1 that are key amino acids for recognition by neutralizing MAb NG8 and further help define a region in CNF1 that is critical for full toxin binding to HEp-2 cells.Cytotoxic necrotizing factor type 1 (CNF1), a toxin made by many uropathogenic and other extraintestinal isolates of Escherichia coli, is a 115-kDa member of the Rho family of GTPaseactivating toxins (for reviews, see references 2, 3, 6, 19, and 26). CNF1 deamidates glutamine 63 (Q63) of RhoA and glutamine 61 (Q61) of Rac1 and Cdc42; these modifications lead to constitutive activation of the target GTPases (1), which in turn results in downstream effects on the mammalian cell phenotype and cell cycle. The typical phenotype associated with CNF1 intoxication in cell cultures is multinucleation, as has been reported previously for HEp-2 cells (14, 15), HeLa cells (13), and Swiss 3T3 cells (23). In addition, this toxin can be cytotoxic to cell lines such as 5637 human bladder cells (25). CNF1-intoxicated cells may also display formation of stress fibers, focal adhesions, lamellipodia, and filopodia and rearrangement of the actin cytoskeleton (16,20,30). In vivo, CNF1 evokes necrosis when it is injected intradermally into rabbits (9). The contribution of CNF1 to uropathogenic E. coli virulence has also been demonstrated using a rat model of acute prostatitis (28), as well as a mouse model of ascending urinary tract infection (11,29,33). In the latter model, intraurethral infection with a CNF1-positive strain leads to an increase...

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High yield purification and first structural characterization of the full‐length bacterial toxin CNF1

Colarusso

Caterino

Fabbri

et al. 2017

Biotechnology Progress

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The Cytotoxic Necrotizing Factor 1 (CNF1) is a bacterial toxin secreted by certain Escherichia coli strains causing severe pathologies, making it a protein of pivotal interest in toxicology. In parallel, the CNF1 capability to influence important neuronal processes, like neuronal arborization, astrocytic support, and efficient ATP production, has been efficiently used in the treatment of neurological diseases, making it a promising candidate for therapy. Nonetheless, there are still some unsolved issues about the CNF1 mechanism of action and structuration probably caused by the difficulty to achieve sufficient amounts of the full-length protein for further studies. Here, we propose an efficient strategy for the production and purification of this toxin as a his-tagged recombinant protein from E. coli extracts (CNF1-H8). CNF1-H8 was expressed at the low temperature of 15°C to diminish its characteristic degradation. Then, its purification was achieved using an immobilized metal affinity chromatography (IMAC) and a size exclusion chromatography so as to collect up to 8 mg of protein per liter of culture in a highly pure form. Routine dynamic light scattering (DLS) experiments showed that the recombinant protein preparations were homogeneous and preserved this state for a long time. Furthermore, CNF1-H8 functionality was confirmed by testing its activity on purified RhoA and on HEp-2 cultured cells. Finally, a first structural characterization of the full-length toxin in terms of secondary structure and thermal stability was performed by circular dichroism (CD). These studies demonstrate that our system can be used to produce high quantities of pure recombinant protein for a detailed structural analysis. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:150-159, 2018.

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Two Domains of Cytotoxic Necrotizing Factor Type 1 Bind the Cellular Receptor, Laminin Receptor Precursor Protein

Cited by 29 publications

References 34 publications

Laminin receptor initiates bacterial contact with the blood brain barrier in experimental meningitis models

Laminin receptor initiates bacterial contact with the blood brain barrier in experimental meningitis models

Cytotoxic Necrotizing Factor Type 1-Neutralizing Monoclonal Antibody NG8 Recognizes Three Amino Acids in a C-Terminal Region of the Toxin and Reduces Toxin Binding to HEp-2 Cells

High yield purification and first structural characterization of the full‐length bacterial toxin CNF1

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