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Insertion-trigger residues differentially modulate endosomal escape by cytotoxic necrotizing factor toxins

2021; Elsevier BV; Volume: 297; Issue: 5 Linguagem: Inglês

10.1016/j.jbc.2021.101347

ISSN

1083-351X

Autores

Elizabeth E. Haywood, Nicholas B. Handy, James W. Lopez, Mengfei Ho, Brenda A. Wilson,

Tópico(s)

Venomous Animal Envenomation and Studies

Resumo

The cellular specificity, potency, and modular nature of bacterial protein toxins enable their application for targeted cytosolic delivery of therapeutic cargo. Efficient endosomal escape is a critical step in the design of bacterial toxin-inspired drug delivery (BTIDD) vehicles to avoid lysosomal degradation and promote optimal cargo delivery. The cytotoxic necrotizing factor (CNF) family of modular toxins represents a useful model for investigating cargo-delivery mechanisms due to the availability of many homologs with high sequence identity, their flexibility in swapping domains, and their differential activity profiles. Previously, we found that CNFy is more sensitive to endosomal acidification inhibitors than CNF1 and CNF2. Here, we report that CNF3 is even less sensitive than CNF1/2. We identified two amino acid residues within the putative translocation domain (E374 and E412 in CNFy, Q373 and S411 in CNF3) that differentiate between these two toxins. Swapping these corresponding residues in each toxin changed the sensitivity to endosomal acidification and efficiency of cargo-delivery to be more similar to the other toxin. Results suggested that trafficking to the more acidic late endosome is required for cargo delivery by CNFy but not CNF3. This model was supported by results from toxin treatment of cells in the presence of NH4Cl, which blocks endosomal acidification, and of small-molecule inhibitors EGA, which blocks trafficking to late endosomes, and ABMA, which blocks endosomal escape and trafficking to the lysosomal degradative pathway. These findings suggest that it is possible to fine-tune endosomal escape and cytosolic cargo delivery efficiency in designing BTIDD platforms. The cellular specificity, potency, and modular nature of bacterial protein toxins enable their application for targeted cytosolic delivery of therapeutic cargo. Efficient endosomal escape is a critical step in the design of bacterial toxin-inspired drug delivery (BTIDD) vehicles to avoid lysosomal degradation and promote optimal cargo delivery. The cytotoxic necrotizing factor (CNF) family of modular toxins represents a useful model for investigating cargo-delivery mechanisms due to the availability of many homologs with high sequence identity, their flexibility in swapping domains, and their differential activity profiles. Previously, we found that CNFy is more sensitive to endosomal acidification inhibitors than CNF1 and CNF2. Here, we report that CNF3 is even less sensitive than CNF1/2. We identified two amino acid residues within the putative translocation domain (E374 and E412 in CNFy, Q373 and S411 in CNF3) that differentiate between these two toxins. Swapping these corresponding residues in each toxin changed the sensitivity to endosomal acidification and efficiency of cargo-delivery to be more similar to the other toxin. Results suggested that trafficking to the more acidic late endosome is required for cargo delivery by CNFy but not CNF3. This model was supported by results from toxin treatment of cells in the presence of NH4Cl, which blocks endosomal acidification, and of small-molecule inhibitors EGA, which blocks trafficking to late endosomes, and ABMA, which blocks endosomal escape and trafficking to the lysosomal degradative pathway. These findings suggest that it is possible to fine-tune endosomal escape and cytosolic cargo delivery efficiency in designing BTIDD platforms. Modular bacterial toxins deliver their catalytic cargo into the cytosol of specific target cells. After binding and cellular uptake, these toxins transport their toxic cargo to the cytosol through multiple trafficking pathways, the most common of which involve retrograde transport through the endoplasmic reticulum or endocytic trafficking from early to late endosomes followed by pH-dependent endosomal escape. Already, a number of modular toxins have been exploited for their ability to deliver heterologous cargo molecules to the cytosol, including fluorescent proteins (1Ho M. Chang L.H. Pires-Alves M. Thyagarajan B. Bloom J.E. Gu Z. Aberle K.K. Teymorian S.A. Bannai Y. Johnson S.C. McArdle J.J. Wilson B.A. Recombinant botulinum neurotoxin A heavy chain-based delivery vehicles for neuronal cell targeting.Protein Eng. Des. Sel. 2011; 24: 247-253Crossref PubMed Scopus (18) Google Scholar), epitope tags (2Fayolle C. Osickova A. Osicka R. Henry T. Rojas M.J. Saron M.F. Sebo P. Leclerc C. Delivery of multiple epitopes by recombinant detoxified adenylate cyclase of Bordetella pertussis induces protective antiviral immunity.J. Virol. 2001; 75: 7330-7338Crossref PubMed Scopus (59) Google Scholar), nanobodies (3McNutt P.M. Vazquez-Cintron E.J. Tenezaca L. Ondeck C.A. Kelly K.E. Mangkhalakhili M. Machamer J.B. Angeles C.A. Glotfelty E.J. Cika J. Benjumea C.H. Whitfield J.T. Band P.A. Shoemaker C.B. 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Shoichet M.S. Attenuated diphtheria toxin mediates siRNA delivery.Sci. Adv. 2020; 6eaaz4848Crossref PubMed Scopus (8) Google Scholar, 12Facchini L.M. Lingwood C.A. A verotoxin 1 B subunit-lambda CRO chimeric protein specifically binds both DNA and globotriaosylceramide (Gb(3)) to effect nuclear targeting of exogenous DNA in Gb(3) positive cells.Exp. Cell Res. 2001; 269: 117-129Crossref PubMed Scopus (20) Google Scholar, 13Lu Z. Paolella B.R. Truex N.L. Loftis A.R. Liao X. Rabideau A.E. Brown M.S. Busanovich J. Beroukhim R. Pentelute B.L. Targeting cancer gene dependencies with anthrax-mediated delivery of peptide nucleic acids.ACS Chem. Biol. 2020; 15: 1358-1369Crossref PubMed Scopus (7) Google Scholar). Bacterial toxin-inspired drug delivery (BTIDD) platforms, such as those described for the cytotoxic necrotizing factor (CNF) toxins (14Haywood E.E. Ho M. Wilson B.A. Modular domain swapping among the bacterial cytotoxic necrotizing factor (CNF) family for efficient cargo delivery into mammalian cells.J. Biol. Chem. 2018; 293: 3860-3870Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar) that assemble from modular components, could be expanded to noncognate therapeutic cargos if the determinants for efficient cytosolic delivery of the biologic cargo were more fully understood. CNF toxins are Rho-deamidating toxins that access their cytosolic targets through trafficking to and escape from acidified endosomes (15Knust Z. Schmidt G. Cytotoxic necrotizing factors (CNFs) – a growing toxin family.Toxins (Basel). 2010; 2: 116-127Crossref PubMed Scopus (35) Google Scholar). The CNF toxin family is comprised of at least nine full-length homologs sharing 54 to 84% identity (14Haywood E.E. Ho M. Wilson B.A. Modular domain swapping among the bacterial cytotoxic necrotizing factor (CNF) family for efficient cargo delivery into mammalian cells.J. Biol. Chem. 2018; 293: 3860-3870Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar), with the highest identity being shared by CNF1 and CNF2 at 84%. The high sequence identities, yet distinct cellular activities, observed among the CNF toxin family members enable probing for discriminatory determinants that modulate the cargo-delivery process. For example, previous investigation of four toxins from this family (CNF1, CNF2, and CNF3 from Escherichia coli and CNFy from Yersinia pseudotuberculosis) revealed differences in cargo-delivery efficiency and compatibility of intertoxin domain assembly among these four toxins (14Haywood E.E. Ho M. Wilson B.A. Modular domain swapping among the bacterial cytotoxic necrotizing factor (CNF) family for efficient cargo delivery into mammalian cells.J. Biol. Chem. 2018; 293: 3860-3870Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). A recent crystal structure of CNFy revealed five structural domains (16Chaoprasid P. Lukat P. Muhlen S. Heidler T. Gazdag E.M. Dong S. Bi W. Ruter C. Kirchenwitz M. Steffen A. Jansch L. Stradal T.E.B. Dersch P. Blankenfeldt W. Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication.EMBO J. 2021; 40e105202Crossref PubMed Scopus (4) Google Scholar). The cellular receptor-binding domain is located near the N-terminus (CNFy residues 23–134). For CNF1 and CNF2, this domain binds laminin precursor receptor (LPR) (17Kim K.J. Chung J.W. Kim K.S. 67-kDa laminin receptor promotes internalization of cytotoxic necrotizing factor 1-expressing Escherichia coli K1 into human brain microvascular endothelial cells.J. Biol. Chem. 2005; 280: 1360-1368Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 18McNichol B.A. Rasmussen S.B. Carvalho H.M. Meysick K.C. O'Brien A.D. Two domains of cytotoxic necrotizing factor type 1 bind the cellular receptor, laminin receptor precursor protein.Infect. Immun. 2007; 75: 5095-5104Crossref PubMed Scopus (23) Google Scholar), while the cellular receptors for the other CNF toxins have not been established. Although catalytically inactive CNFy has been shown to retard entry of CNF1 into cells, CNFy does not bind LPR, suggesting an overlapping coreceptor (19Blumenthal B. Hoffmann C. Aktories K. Backert S. Schmidt G. The cytotoxic necrotizing factors from Yersinia pseudotuberculosis and from Escherichia coli bind to different cellular receptors but take the same route to the cytosol.Infect. Immun. 2007; 75: 3344-3353Crossref PubMed Scopus (45) Google Scholar). CNF1 and CNFy reportedly have an additional binding region in the C-terminus: CNF1 residues 709 to 730 bind to Lu/BCAM adhesion molecule (20Piteau M. Papatheodorou P. Schwan C. Schlosser A. Aktories K. Schmidt G. Lu/BCAM adhesion glycoprotein is a receptor for Escherichia coli cytotoxic necrotizing factor 1 (CNF1).PLoS Pathog. 2014; 10e1003884Crossref PubMed Scopus (24) Google Scholar), and CNFy residues 772 to 779 bind to heparan sulfates (21Kowarschik S. Schollkopf J. Muller T. Tian S. Knerr J. Bakker H. Rein S. Dong M. Weber S. Grosse R. Schmidt G. Yersinia pseudotuberculosis cytotoxic necrotizing factor interacts with glycosaminoglycans.FASEB J. 2021; 35e21647Crossref PubMed Scopus (2) Google Scholar). Based on previously predicted functional domain organization of the CNF family (22Knust Z. Blumenthal B. Aktories K. Schmidt G. Cleavage of Escherichia coli cytotoxic necrotizing factor 1 is required for full biologic activity.Infect. Immun. 2009; 77: 1835-1841Crossref PubMed Scopus (31) Google Scholar, 23Lemichez E. Flatau G. Bruzzone M. Boquet P. Gauthier M. Molecular localization of the Escherichia coli cytotoxic necrotizing factor CNF1 cell-binding and catalytic domains.Mol. Microbiol. 1997; 24: 1061-1070Crossref PubMed Scopus (117) Google Scholar, 24Pei S. Doye A. Boquet P. Mutation of specific acidic residues of the CNF1 T domain into lysine alters cell membrane translocation of the toxin.Mol. Microbiol. 2001; 41: 1237-1247Crossref PubMed Scopus (51) Google Scholar), the N-terminal membrane translocation module, comprised of domains D1 and D3 (CNFy residues 135–530), facilitates endosomal escape of the C-terminal cargo, which includes domain D4 of unknown function (CNFy residues 530–700) and the catalytic Rho-deamidase domain D5 (CNFy residues 718–1014) (16Chaoprasid P. Lukat P. Muhlen S. Heidler T. Gazdag E.M. Dong S. Bi W. Ruter C. Kirchenwitz M. Steffen A. Jansch L. Stradal T.E.B. Dersch P. Blankenfeldt W. Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication.EMBO J. 2021; 40e105202Crossref PubMed Scopus (4) Google Scholar), which is also consistent with the structure of the catalytic domain of CNF1 (25Buetow L. Flatau G. Chiu K. Boquet P. Ghosh P. Structure of the Rho-activating domain of Escherichia coli cytotoxic necrotizing factor 1.Nat. Struct. Biol. 2001; 8: 584-588Crossref PubMed Scopus (86) Google Scholar). A key step in the cellular intoxication process of CNF toxins involves a pH-dependent membrane insertion that occurs in an acidic endosome (26Contamin S. Galmiche A. Doye A. Flatau G. Benmerah A. Boquet P. The p21 Rho-activating toxin cytotoxic necrotizing factor 1 is endocytosed by a clathrin-independent mechanism and enters the cytosol by an acidic-dependent membrane translocation step.Mol. Biol. Cell. 2000; 11: 1775-1787Crossref PubMed Scopus (78) Google Scholar). Based on comparative sequence analysis that predicted a similar organization in the translocation region of CNF1 to the so-called "dagger" membrane-insertion motif (helices TH8–TH9) found in the T domain of diphtheria toxin (DT) (27Choe S. Bennett M.J. Fujii G. Curmi P.M. Kantardjieff K.A. Collier R.J. Eisenberg D. The crystal structure of diphtheria toxin.Nature. 1992; 357: 216-222Crossref PubMed Scopus (562) Google Scholar, 28Senzel L. Gordon M. Blaustein R.O. Oh K.J. Collier R.J. Finkelstein A. Topography of diphtheria toxin's T domain in the open channel state.J. Gen. Physiol. 2000; 115: 421-434Crossref PubMed Scopus (61) Google Scholar), a model for the pH-dependent insertion step of the cargo-delivery process was previously proposed involving a putative helix-loop-helix (HLH) region (residues 350–412 in CNF1) (24Pei S. Doye A. Boquet P. Mutation of specific acidic residues of the CNF1 T domain into lysine alters cell membrane translocation of the toxin.Mol. Microbiol. 2001; 41: 1237-1247Crossref PubMed Scopus (51) Google Scholar). This putative HLH of CNF1 contains four highly conserved acidic residues (D373, D379, E382, E383) in the postulated loop region that were proposed to become protonated in the acidic environment of the late endosome, thereby allowing insertion as a "dagger" into the membrane. Once on the cytosolic side of the membrane, the putative HLH would again become deprotonated, locking the HLH in place and initiating the membrane translocation process and cytosolic delivery of the cargo. However, although these four acidic residues were confirmed to be important for cargo delivery of CNF1 (24Pei S. Doye A. Boquet P. Mutation of specific acidic residues of the CNF1 T domain into lysine alters cell membrane translocation of the toxin.Mol. Microbiol. 2001; 41: 1237-1247Crossref PubMed Scopus (51) Google Scholar), the crystal structure of CNFy (16Chaoprasid P. Lukat P. Muhlen S. Heidler T. Gazdag E.M. Dong S. Bi W. Ruter C. Kirchenwitz M. Steffen A. Jansch L. Stradal T.E.B. Dersch P. Blankenfeldt W. Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication.EMBO J. 2021; 40e105202Crossref PubMed Scopus (4) Google Scholar) did not reveal the predicted HLH structure, and it was proposed that since the structure was determined under neutral conditions, perhaps the region changes its conformation once it is in an acidic environment. Other studies have revealed that there are differences among the CNF toxins with regard to efficiency (14Haywood E.E. Ho M. Wilson B.A. Modular domain swapping among the bacterial cytotoxic necrotizing factor (CNF) family for efficient cargo delivery into mammalian cells.J. Biol. Chem. 2018; 293: 3860-3870Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar) and pH dependency (29Repella T.L. Ho M. Wilson B.A. Determinants of pH-dependent modulation of translocation in dermonecrotic G-protein-deamidating toxins.Toxins (Basel). 2013; 5: 1167-1179Crossref PubMed Scopus (5) Google Scholar) of cargo delivery. CNF1, CNF2, and CNFy toxins have differential dose-dependent responses to inhibitors of endosomal acidification (29Repella T.L. Ho M. Wilson B.A. Determinants of pH-dependent modulation of translocation in dermonecrotic G-protein-deamidating toxins.Toxins (Basel). 2013; 5: 1167-1179Crossref PubMed Scopus (5) Google Scholar), such as NH4Cl that acts as a weak base to raise the endosomal pH and bafilomycin A1 that blocks acidification by inhibition of the vacuolar ATPase proton pump. These findings suggest that there may be other protein determinants besides the four conserved acidic residues that dictate pH sensitivity and influence CNF toxin cargo delivery efficiency. In addition to NH4Cl and bafilomycin A1, two other small-molecule inhibitors of cellular trafficking pathways, 4-bromo-benzaldehyde N-(2,6-dimethylphenyl) semi-carbazone (EGA) and 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA), have been used to investigate intoxication mechanisms of modular protein toxins that traffic through acidified endosomes to deliver their cargo into the cytosol. EGA blocks trafficking from the early endosome to the late endosome (30Azarnia Tehran D. Zanetti G. Leka O. Lista F. Fillo S. Binz T. Shone C.C. Rossetto O. Montecucco C. Paradisi C. Mattarei A. Pirazzini M. A novel inhibitor prevents the peripheral neuroparalysis of botulinum neurotoxins.Sci. Rep. 2015; 5: 17513Crossref PubMed Scopus (21) Google Scholar, 31Gillespie E.J. Ho C.L. Balaji K. Clemens D.L. Deng G. Wang Y.E. Elsaesser H.J. Tamilselvam B. Gargi A. Dixon S.D. France B. Chamberlain B.T. Blanke S.R. Cheng G. de la Torre J.C. et al.Selective inhibitor of endosomal trafficking pathways exploited by multiple toxins and viruses.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E4904-4912Crossref PubMed Scopus (58) Google Scholar, 32Schnell L. Mittler A.K. Sadi M. Popoff M.R. Schwan C. Aktories K. Mattarei A. Azarnia Tehran D. Montecucco C. Barth H. EGA protects mammalian cells from Clostridium difficile CDT, Clostridium perfringens iota toxin and Clostridium botulinum C2 toxin.Toxins (Basel). 2016; 8: 101Crossref PubMed Scopus (7) Google Scholar), which prevents some toxins from reaching the lower pH compartment needed for triggering membrane insertion and translocation. In contrast, ABMA reportedly blocks intoxication independent of endosomal acidification and at a stage after acidification (33Wu Y. Pons V. Goudet A. Panigai L. Fischer A. Herweg J.A. Kali S. Davey R.A. Laporte J. Bouclier C. Yousfi R. Aubenque C. Merer G. Gobbo E. Lopez R. et al.ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments.Sci. Rep. 2017; 7: 15567Crossref PubMed Scopus (8) Google Scholar) and also inhibits trafficking from the late endosome to the lysosomal degradation pathway (34Wu Y. Boulogne C. Carle S. Podinovskaia M. Barth H. Spang A. Cintrat J.C. Gillet D. Barbier J. Regulation of endo-lysosomal pathway and autophagic flux by broad-spectrum antipathogen inhibitor ABMA.FEBS J. 2020; 287: 3184-3199Crossref PubMed Scopus (6) Google Scholar). Thus, through use of NH4Cl, EGA, and ABMA, it may be possible to identify key points along the endosomal pathway that may differentiate among the CNF toxins: acidification, trafficking from the early endosome to the late endosome, trafficking from the late endosome to the lysosome, and/or escape of the cargo from the endosome. Here, we compared CNF1, CNF2, CNF3, and CNFy for their sensitivity to inhibitors of endosomal acidification and trafficking using cell-based SRE-luciferase assays, performed as previously described (14Haywood E.E. Ho M. Wilson B.A. Modular domain swapping among the bacterial cytotoxic necrotizing factor (CNF) family for efficient cargo delivery into mammalian cells.J. Biol. Chem. 2018; 293: 3860-3870Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar, 29Repella T.L. Ho M. Wilson B.A. Determinants of pH-dependent modulation of translocation in dermonecrotic G-protein-deamidating toxins.Toxins (Basel). 2013; 5: 1167-1179Crossref PubMed Scopus (5) Google Scholar). We found that among the CNF toxins, CNF3 was the most tolerant to inhibition of endosomal acidification, while CNFy was the most sensitive. To identify protein determinants that respond to changes in endosomal pH and to differentiate among the toxins, we generated and characterized a series of chimeric toxins between CNF3 and CNFy and identified the putative HLH region of the translocation domain as the region responsible for discriminating their pH sensitivities. Site-specific mutational analysis identified two acidic residues within this region responsible for mediating the differential sensitivities to NH4Cl. CNF3 and CNFy were also investigated for their differential sensitivity to EGA or ABMA, enabling discrimination of the exit points taken by these toxins in the intoxication pathway. It has been established previously that agents that raise endosomal pH such as NH4Cl antagonize the entry of CNF toxin cargos into the cytosol (26Contamin S. Galmiche A. Doye A. Flatau G. Benmerah A. Boquet P. The p21 Rho-activating toxin cytotoxic necrotizing factor 1 is endocytosed by a clathrin-independent mechanism and enters the cytosol by an acidic-dependent membrane translocation step.Mol. Biol. Cell. 2000; 11: 1775-1787Crossref PubMed Scopus (78) Google Scholar, 29Repella T.L. Ho M. Wilson B.A. Determinants of pH-dependent modulation of translocation in dermonecrotic G-protein-deamidating toxins.Toxins (Basel). 2013; 5: 1167-1179Crossref PubMed Scopus (5) Google Scholar). NH4Cl chemically counteracts the acidification of the endosome, preventing the low pH necessary for translocation of toxin cargos. Previous studies showed that CNF1, CNF2, and CNFy had differential sensitivities to NH4Cl (29Repella T.L. Ho M. Wilson B.A. Determinants of pH-dependent modulation of translocation in dermonecrotic G-protein-deamidating toxins.Toxins (Basel). 2013; 5: 1167-1179Crossref PubMed Scopus (5) Google Scholar), while CNF3 sensitivity to NH4Cl was not tested previously. We first compared the activities of wild-type CNF1, CNF2, CNF3, and CNFy toxins in a cell-based SRE-luciferase assay in response to NH4Cl. As shown in Figure 1, pretreatment of HEK293T cells with NH4Cl blocked the intracellular activity of all four toxins in a dose-dependent manner. In addition to exhibiting differential cargo delivery efficiencies, as previously described (14Haywood E.E. Ho M. Wilson B.A. Modular domain swapping among the bacterial cytotoxic necrotizing factor (CNF) family for efficient cargo delivery into mammalian cells.J. Biol. Chem. 2018; 293: 3860-3870Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar), all four of the CNF toxins displayed differential sensitivities to inhibition of endosomal acidification at a uniform toxin dose of 100 ng/ml (Fig. 1A). Consistent with previously described studies for CNF1, CNF2 and CNFy (29Repella T.L. Ho M. Wilson B.A. Determinants of pH-dependent modulation of translocation in dermonecrotic G-protein-deamidating toxins.Toxins (Basel). 2013; 5: 1167-1179Crossref PubMed Scopus (5) Google Scholar), CNFy was the most sensitive to NH4Cl, CNF2 had intermediate sensitivity, and CNF1 and CNF3 were the most resistant. As was noted previously (14Haywood E.E. Ho M. Wilson B.A. Modular domain swapping among the bacterial cytotoxic necrotizing factor (CNF) family for efficient cargo delivery into mammalian cells.J. Biol. Chem. 2018; 293: 3860-3870Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar), it is important to minimize the effects of differences in substrate specificities and receptor-mediated uptake when comparing CNF toxins with each other. Thus, the NH4Cl-sensitivity assay was also performed at toxin concentrations equivalent to their respective EC50 values, where the limiting step for the observed toxin activity equates to their efficiency of cargo delivery, such that differences in receptor-binding efficiencies were minimized (Fig. 1B). In this case, CNFy remained the most sensitive to NH4Cl treatment, requiring only 5 mM NH4Cl to block all CNFy-mediated reporter activation, whereas CNF3 remained the least sensitive. However, when tested at their respective EC50 values, CNF1 and CNF2 displayed similar intermediate sensitivities (Fig. 1B). This finding is consistent with the fact that these two toxins have the greatest sequence homology and are identical regarding charged residues in the previously identified HLH insertion region. Overall, these results suggested that CNF3 and CNFy represent the CNF toxins with the most and least sensitivity to endosomal acidification, respectively, and thus we asked whether we could identify specific protein determinants that drive this observed difference in sensitivity between them. To identify the determinants that discriminate CNF toxin sensitivity to inhibition of endosomal acidification, we applied a binary search approach to define the region of interest (i.e., the region that mediates the differential responses) and then selected new joining sites within that region to further refine the search. We generated a series of chimeric proteins with the N-terminal delivery domain of the least sensitive toxin CNF3 and the cargo domain of the most sensitive toxin CNFy (CNF3y) (Fig. 2A) and compared their sensitivities to NH4Cl inhibition in cell-based activity reporter assays, as described above. Accordingly, the chimera CNF3y-223 was joined downstream of the putative N-terminal receptor-binding domain (residues 23–134) (18McNichol B.A. Rasmussen S.B. Carvalho H.M. Meysick K.C. O'Brien A.D. Two domains of cytotoxic necrotizing factor type 1 bind the cellular receptor, laminin receptor precursor protein.Infect. Immun. 2007; 75: 5095-5104Crossref PubMed Scopus (23) Google Scholar, 35Fabbri A. Gauthier M. Boquet P. The 5' region of cnf1 harbours a translational regulatory mechanism for CNF1 synthesis and encodes the cell-binding domain of the toxin.Mol. Microbiol. 1999; 33: 108-118Crossref PubMed Scopus (35) Google Scholar), while CNF3y-519 was joined upstream of the suspected cleavage site (residues 532–544) that defines the putative cargo and delivery vehicle domains (22Knust Z. Blumenthal B. Aktories K. Schmidt G. Cleavage of Escherichia coli cytotoxic necrotizing factor 1 is required for full biologic activity.Infect. Immun. 2009; 77: 1835-1841Crossref PubMed Scopus (31) Google Scholar). As shown in Figure 2B, CNF3y-223 was completely inhibited by 5 mM NH4Cl, matching the response observed for CNFy, while the inhibitor profile of CNF3y-519 resembled that of CNF3. These results confirmed that the region differentially sensing pH is located within the putative translocation domain (residues 223–519). To minimize structural perturbations, additional joining sites at highly conserved positions 317 and 428 within the newly defined pH-sensing region were next explored, based on amino acid sequence alignment, secondary structure predictions, and regional pI calculations (data not shown). The resulting chimeric toxins CNF3y-317 and CNF3y-428, respectively, were generated and characterized using the SRE-luciferase assay. As shown in Figure 2C, the inhibitor profiles showed that CNF3y-317 is as sensitive to NH4Cl as CNFy, while CNF3y-428 is as tolerant as CNF3, thereby narrowing the pH-sensing region to positions 317 to 428, which includes the putative HLH region and the previously identified acidic residues in CNF1 (D373, D379, E382, and E383) that are important for translocation (24Pei S. Doye A. Boquet P. Mutation of specific acidic residues of the CNF1 T domain into lysine alters cell membrane translocation of the toxin.Mol. Microbiol. 2001; 41: 1237-1247Crossref PubMed Scopus (51) Google Scholar) and are conserved for all known CNF toxins. To further identify the residues within the putative HLH region that contribute to the differences between CNF3 and CNFy in sensing pH changes, three new chimeric toxins were constructed: CNF3y-349, CNF3y-375, and CNF3y-412. The resulting inhibitor profiles showed that CNF3y-349 like CNFy is more sensitive to NH4Cl, while CNF3y-412 like CNF3 is more tolerant. Interestingly, the chimera CNF3y-375 is intermediate in sensitivity to NH4Cl, suggesting that one or more residues within each of the regions flanking the joining site at position 375 influence the response of the chimeric toxin to endosomal acidification. Moreover, chimera CNF3y-412 has a lower EC50 value of 0.056 nM, compared with CNF3y-349 and CNF3y-375, each with EC50 values of 0.28 nM and 0.20 nM, respectively (Table 1), further supporting the importance of this region in determining the efficiency of cargo delivery.Table 1EC50 values of wild-type and mutant CNF toxinsaThe EC50 values were calculated from dose–response

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