Artigo Acesso aberto Revisado por pares

The Role of Electrostatics in Colicin Nuclease Domain Translocation into Bacterial Cells

2007; Elsevier BV; Volume: 282; Issue: 43 Linguagem: Inglês

10.1074/jbc.m705883200

ISSN

1083-351X

Autores

Daniel Walker, Khédidja Mosbahi, Mireille Vankemmelbeke, Richard James, Colin Kleanthous,

Tópico(s)

Escherichia coli research studies

Resumo

The mechanism(s) by which nuclease colicins translocate distinct cytotoxic enzymes (DNases, rRNases, and tRNases) to the cytoplasm of Escherichia coli is unknown. Previous in vitro investigations on isolated colicin nuclease domains have shown that they have a strong propensity to associate with anionic phospholipid vesicles, implying that electrostatic interactions with biological membranes play a role in their import. In the present work we set out to test this hypothesis in vivo. We show that cell killing by the DNase toxin colicin E9 of E. coli HDL11, a strain in which the level of anionic phospholipid and hence inner membrane charge is regulated by isopropyl β-d-thiogalactopyranoside induction, is critically dependent on the level of inducer, whereas this is not the case for pore-forming colicins that take the same basic route into the periplasm. Moreover, there is a strong correlation between the level and rate of HDL11 cell killing and the net positive charge on a colicin DNase, with similar effects seen for wild type E. coli cells, data that are consistent with a direct, electrostatically mediated interaction between colicin nucleases and the bacterial inner membrane. We next sought to identify how membrane-associated colicin nucleases might be translocated into the cell. We show that neither the Sec or Tat systems are involved in nuclease colicin uptake but that nuclease colicin toxicity is instead dependent on functional FtsH, an inner membrane AAA+ ATPase and protease that dislocates misfolded membrane proteins to the cytoplasm for destruction. The mechanism(s) by which nuclease colicins translocate distinct cytotoxic enzymes (DNases, rRNases, and tRNases) to the cytoplasm of Escherichia coli is unknown. Previous in vitro investigations on isolated colicin nuclease domains have shown that they have a strong propensity to associate with anionic phospholipid vesicles, implying that electrostatic interactions with biological membranes play a role in their import. In the present work we set out to test this hypothesis in vivo. We show that cell killing by the DNase toxin colicin E9 of E. coli HDL11, a strain in which the level of anionic phospholipid and hence inner membrane charge is regulated by isopropyl β-d-thiogalactopyranoside induction, is critically dependent on the level of inducer, whereas this is not the case for pore-forming colicins that take the same basic route into the periplasm. Moreover, there is a strong correlation between the level and rate of HDL11 cell killing and the net positive charge on a colicin DNase, with similar effects seen for wild type E. coli cells, data that are consistent with a direct, electrostatically mediated interaction between colicin nucleases and the bacterial inner membrane. We next sought to identify how membrane-associated colicin nucleases might be translocated into the cell. We show that neither the Sec or Tat systems are involved in nuclease colicin uptake but that nuclease colicin toxicity is instead dependent on functional FtsH, an inner membrane AAA+ ATPase and protease that dislocates misfolded membrane proteins to the cytoplasm for destruction. Colicins are SOS-induced protein antibiotics that penetrate and kill cells in competing Escherichia coli populations (1Kirkup B.C. Kim M.A. Nature. 2004; 428: 412-414Crossref PubMed Scopus (352) Google Scholar, 2Czaran T.L. Kim R.F. Pagie L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 786-790Crossref PubMed Scopus (371) Google Scholar). Colicin entry is known to begin with binding to an extracellular receptor followed by translocation across the outer membrane (OM) 3The abbreviations used are: OM, outer membrane; IM, inner membrane; IPTG, isopropyl β-d-thiogalactopyranoside; ER, endoplasmic reticulum; LPS, lipopolysaccharide.3The abbreviations used are: OM, outer membrane; IM, inner membrane; IPTG, isopropyl β-d-thiogalactopyranoside; ER, endoplasmic reticulum; LPS, lipopolysaccharide. by a mechanism that has similarities to that used by filamentous bacteriophages in the early stages of phage infection (3Cascales E. Kim S.K. Duché D. Kleanthous C. Lloubès R. Postle K. Riley M. Slatin S. Cavard D. Microbiol. Mol. Biol. Rev. 2007; 71: 158-229Crossref PubMed Scopus (756) Google Scholar). Once in the periplasm the cytotoxic domains of colicins target the IM or, in the case of the nuclease colicins, are translocated entirely across the IM to reach their cytoplasmic nucleic acid substrates (3Cascales E. Kim S.K. Duché D. Kleanthous C. Lloubès R. Postle K. Riley M. Slatin S. Cavard D. Microbiol. Mol. Biol. Rev. 2007; 71: 158-229Crossref PubMed Scopus (756) Google Scholar, 4James R. Kim C.N. Moore G.R. Kleanthous C. Biochimie (Paris). 2002; 84: 381-389Crossref PubMed Scopus (76) Google Scholar). There is currently no information available as to how this latter step is accomplished, although it is likely to require unfolding of the nuclease (5Mosbahi K. Kim C. Keeble A.H. Mobasheri H. Morel B. James R. Moore G.R. Lea E.J. Kleanthous C. Nat. Struct. Biol. 2002; 9: 476-484Crossref PubMed Scopus (44) Google Scholar). Passage across the OM for both pore-forming and nuclease type colicins is mediated by an OM receptor(s) and proteins of either the Tol (for Group A colicins such as A, N, and E1–E9) or Ton (for Group B colicins such as B, D, Ia, and Ib) complexes. Colicins are able to utilize a wide variety of OM proteins as their primary receptor, such as FepA, FhuA, Cir, BtuB, and OmpF, all involved in passive or active nutrient transport across the OM (3Cascales E. Kim S.K. Duché D. Kleanthous C. Lloubès R. Postle K. Riley M. Slatin S. Cavard D. Microbiol. Mol. Biol. Rev. 2007; 71: 158-229Crossref PubMed Scopus (756) Google Scholar). In contrast, the translocation step is restricted to either the Ton or Tol proteins, with movement across the OM likely by a common mechanism because colicins can be engineered to take either route by the simple exchange of colicin domains or indeed the requisite phage domains (6Jakes K.S. Kim N.G. Zinder N.D. J. Bacteriol. 1988; 170: 4231-4238Crossref PubMed Google Scholar, 7Braun V. FEMS Microbiol. Rev. 1995; 4: 295-307Crossref Scopus (281) Google Scholar). The physiological role of the Ton (TonB·ExbB·ExbD) complex is in the energy-dependent transport of nutrients across the OM. The complex is coupled to the proton motive force across the IM and is transduced to OM receptors via TonB through so-called "TonB box" sequences, which stimulate the passage of nutrients across the OM (8Wiener M.C. Curr. Opin. Struct. Biol. 2005; 15: 394-400Crossref PubMed Scopus (108) Google Scholar). The Tol·Pal system (TolA·TolQ·TolR·TolB·Pal) is also a transmembrane assembly responsive to the proton-motive force. In contrast to the Ton system, however, the Tol·Pal complex is required for stability of the OM, with recent data suggesting that its physiological role is that of an energized tether that maintains the appropriate juxtaposition between the inner and outer membranes and newly formed peptidoglycan during cell division (9Lloubes R. Kim E. Walburger A. Bouveret E. Lazdunski C. Bernadac A. Journet L. Res. Microbiol. 2001; 152: 523-529Crossref PubMed Scopus (136) Google Scholar, 10Gerding M.A. Kim Y. Pecora N.D. Niki H. de Boer P.A. Mol. Microbiol. 2007; 63: 1008-1025Crossref PubMed Scopus (259) Google Scholar). Association with these energized periplasm-spanning systems bring the cytotoxic domains of pore-forming colicins to the periplasmic side of the IM into which they spontaneously insert forming voltage-dependent ion channels that depolarize the cytoplasmic membrane (11Zakharov S.D. Kim W.A. Biochimie (Paris). 2002; 84: 465-475Crossref PubMed Scopus (26) Google Scholar). In contrast, nuclease type colicins, or at least their cytotoxic domains, must pass into the cytoplasm where they act enzymatically on DNA (colicins E2, E7, E8, and E9), tRNA (E5 and D), or rRNA (E3, E4, and E6) (4James R. Kim C.N. Moore G.R. Kleanthous C. Biochimie (Paris). 2002; 84: 381-389Crossref PubMed Scopus (76) Google Scholar, 12Masaki H. Kim T. Biochimie (Paris). 2002; 84: 433-438Crossref PubMed Scopus (60) Google Scholar). From work on colicins D and E7 has come the suggestion that the nuclease domains are cleaved from the remainder of the toxin while in the periplasm and/or passage across the IM. In the case of colicin D, the protease has been identified as the signal peptidase LepB or a factor processed by LepB (13de Zamaroczy M. Kim L. Lecuyer A. Geli V. Buckingham R.H. Mol. Cell. 2001; 8: 159-168Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 14Shi Z. Kim K.F. Yuan H.S. J. Biol. Chem. 2005; 280: 24663-24668Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). A key consideration for this group of cytotoxins is the absence of any sequence or structural similarity between the different nuclease domains or indeed between nucleases that import via the Tol or Ton pathways, emphasizing that whatever route(s) exist for entry to the cytosol they are insensitive to the structure of the nuclease. Here, we provide the first in vivo evidence demonstrating the importance of nuclease domain charge on colicin translocation, which implies that prior to import to the cytoplasm there is an electrostatically driven association with the E. coli IM. We also identify a putative translocation route for membrane-associated colicin nucleases. Bacterial Strains and Plasmids—The cells were grown in LB broth or on LB agar with kanamycin (50 μg ml-1), tetracycline (5 μg ml-1), chloramphenicol (34 μg ml-1), or ampicillin (100 μg ml-1) where required. AR3289 (W3110 sfhC21 zad220::Tn10) and AR3291 (W3110 ΔftsH3 sfhC21 zad220::Tn10) are described by Tatsuta et al. (15Tatsuta T. Kim T. Bukau B. Kitagawa M. Mori H. Karata K. Ogura T. Mol. Microbiol. 1998; 30: 583-593Crossref PubMed Scopus (96) Google Scholar). JARV15 (MC4100 ΔtatA ΔtatE), BØD (MC4100 ΔtatB), and B1LK0 (ΔtatC) are described by Stanley et al. (16Stanley N.R. Kim K. Berks B.C. Palmer T. J. Bacteriol. 2001; 183: 139-144Crossref PubMed Scopus (140) Google Scholar). GN15 (MC4100 secY205 ompT::kan), GN31 (secY39 ompT::kan), GN32 (secY125 ompT::kan rpsE), NH146 (MC4100 secD1 ompT::kan), and NH195 (secE501) are described by Matsumoto et al. (17Matsumoto G. Kim H. Mori H. Ito K. Genes Cells. 2000; 5: 991-999Crossref PubMed Scopus (23) Google Scholar). Strain HDL11 (pgsA::kan Φ(lacOP-pgsA+)1 lacZ′ lacY::Tn9 lpp2 zdg::Tn10) is described by Kusters et al. (18Kusters R. Kim W. de Kruijff B. J. Biol. Chem. 1991; 266: 8659-8662Abstract Full Text PDF PubMed Google Scholar), and strains SD12 and BW25113, which are wild type for phospholipid biosynthetic enzymes, are described by Shibuya et al. (19Shibuya I. Kim C. Ohta A. J. Bacteriol. 1985; 161: 1086-1092Crossref PubMed Google Scholar) and Baba et al. (20Baba T. Kim T. Hasegawa M. Takai Y. Okumura Y. Baba M. Datsenko K.A. Tomita M. Wanner B.L. Mori H. Mol. Syst. Biol. 2006; 2 (2006.0008)Crossref Scopus (5196) Google Scholar), respectively. The colicin-producing strains BZB2101 (colicin A), BZB2102 (colicin B), BZB2103 (colicin D), BZB2104 (colicin E1), BZB2108 (colicin E5), BZB2114 (colicin Ia), BZB2115 (colicin Ib), PAP1 (colicin M), and BZB2123 (colicin N) are described by Pugsley (21Pugsley A.P. J. Gen. Microbiol. 1985; 131: 369-376PubMed Google Scholar) and were obtained from the Institut Pasteur strain collection. pIFH108 encoding wild type FtsH and derived plasmids encoding the FtsH mutants K201N, F228R, E255Q, D304A, D307A, E418Q, and H421Y have been previously described (22Karata K. Kim T. Wilkinson A.J. Tatsuta T. Ogura T. J. Biol. Chem. 1999; 274: 26225-26232Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 23Karata K. Kim C.S. Wilkinson A.J. Ogura T. Mol. Microbiol. 2001; 39: 890-903Crossref PubMed Scopus (50) Google Scholar, 24Yamada-Inagawa T. Kim T. Karata K. Yamanaka K. Ogura T. J. Biol. Chem. 2003; 278: 50182-50187Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Mutations in the DNase domain of colicin E9 (K21E, K45E, K21E/K45E, S77K/S78R, and S77K/S78R/S80K; DNase numbering) were made by the Stratagene QuikChange method using the plasmid pRJ345 as the template. The NcoI-XhoI fragment of the mutagenized plasmid was ligated into the same sites of the plasmid pCS4. Protein Purification—Nuclease colicins were used either as complexes with their immunity proteins (25Kleanthous C. Kim D. Trends Biochem. Sci. 2000; 26: 624-631Abstract Full Text Full Text PDF Scopus (98) Google Scholar) or as isolated toxins; cell killing kinetics are unaltered by the presence of the immunity protein (26Wallis R. Kim A. Rowe A. Moore G.R. James R. Kleanthous C. Eur. J. Biochem. 1992; 207: 687-695Crossref PubMed Scopus (56) Google Scholar, 27Vankemmelbeke M. Kim B. Moore G.R. Kleanthous C. Penfold C.N. James R. J. Bacteriol. 2005; 187: 4900-4907Crossref PubMed Scopus (23) Google Scholar). The colicin E3·Im3 and colicin E9·Im9 (E2·Im2, E7·Im7, and E8·Im8) complexes and uncomplexed colicin E9 were purified as described previously (28Walker D. Kim G.R. James R. Kleanthous C. Biochemistry. 2003; 42: 4161-4171Crossref PubMed Scopus (47) Google Scholar). Colicin Ia and Ib were purified from strains BZB2114 and BZB2115, respectively (described below). The cells were grown in LB medium (1.6 liters) to an A600 = 0.5, mitomycin C was added to a final concentration of 0.6 μg ml-1, and the cells were grown for a further 4 h all at 37 °C. The harvested cells were resuspended in 30 ml of 50 mm KPi buffer, pH 7.0, and broken by passage through a French press. After removal of the cell debris by centrifugation, ammonium sulfate was added to a final concentration of 114 g liter-1 at 4 °C and stirred for 1 h at this temperature. The precipitate was removed by centrifugation, and ammonium sulfate (193 g liter-1) was added. After centrifugation the supernatant was discarded, and the pellet was resuspended in a small volume of 50 mm KPi, pH 7.0, dialyzed into 2 × 5 liters of the same buffer. The proteins were then applied to a MonoS column and eluted with a 0–300 mm NaCl gradient. Liquid Culture Cell Death Assays—To measure cell death in liquid culture an overnight culture of E. coli HDL11 was diluted 1:100 into 50 ml of LB broth with or without IPTG (20–500 μm) and grown at 37 °C with shaking. After 180 min the appropriate purified colicin was added (A600 =∼0.6). The A600 of each culture was taken at 30-min intervals throughout the experiment. For spot test assays of purified colicin, LB agar plates were overlaid with 5 ml of molten 0.7% (w/v) non-nutrient agar, to which was added 100 μl of a culture of the appropriate indicator strain with an A600 of 0.6–0.8. For AR3291, 500 μl of culture was used, because this strain grows slowly on LB agar. Aliquots of 2 μl of 5-fold dilutions of colicin were spotted on the plates, which were then incubated at 37 °C overnight. To test the cytotoxicity of colicins without prior purification of the protein, colicin expressing cultures were stabbed into LB agar plates and incubated at 37 °C overnight. The plates were exposed to chloroform vapor for 15 min to break the cells and then overlaid with 5 ml of molten 0.7% (w/v) non-nutrient agar containing the indicator strain prepared as described above. The plates were incubated overnight at 37 °C and then inspected for a clear zone of growth inhibition around the test culture. Lux Reporter Assay—All of the assays were done as described previously using the DPD1718, a strain housing an SOS-inducible lux reporter system (27Vankemmelbeke M. Kim B. Moore G.R. Kleanthous C. Penfold C.N. James R. J. Bacteriol. 2005; 187: 4900-4907Crossref PubMed Scopus (23) Google Scholar). DPD1718 cells were grown to mid-log (A ∼ 0.35–0.45), after which they were diluted 1:2 (100 μl total volume) into black 96-well plates with an optical bottom (Nunc), and wild type colicins or mutant proteins were added to a final concentration of 0.4 nm. Trypsin (final concentration, 0.05 mg/ml) was added at regular time intervals between 0 and 30 min after colicin addition. Induction of luminescence was followed over a period of up to 2 h with readings taken every 300 s. Gamma values (27Vankemmelbeke M. Kim B. Moore G.R. Kleanthous C. Penfold C.N. James R. J. Bacteriol. 2005; 187: 4900-4907Crossref PubMed Scopus (23) Google Scholar) for the luminescence induction by each colicin were calculated, and the amount of protection offered by trypsin treatment was obtained by comparing the luminescence induced by colicin in the presence and absence of trypsin. All of the assays were performed at least twice, and error bars, where shown, represent the means ± S.E. for at least two independent experiments. Cell Survival Assay—HDL11 was grown in LB broth at 37 °C to mid-log phase (A600 =∼0.6) in the presence or absence of 100 μm of IPTG. Colicin at a final concentration of 80 nm was added to 1-ml aliquots of the cells with trypsin (1 mg ml-1) added at defined time points after the addition of the colicin. Each sample was left to incubate at 37 °C for a further 30 min, after which cells were diluted in LB and plated onto LB agar with the appropriate antibiotics. After overnight growth at 37 °C, the cells were counted, and the percentage of survival was calculated relative to a control sample to which no colicin was added. Colicin DNase Domain Charge Modulates Cell Killing Efficiency in E. coli Cells Depleted of Anionic Phospholipids—We have demonstrated previously that the colicin E3 rRNase and E9 DNase domains (12 and 15 kDa, respectively) interact with anionic but not neutral phospholipid vesicles, with these electrostatically driven associations causing the domains to become destabilized (29Mosbahi K. Kim D. Lea E. Moore G.R. James R. Kleanthous C. J. Biol. Chem. 2004; 279: 22145-22151Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 30Mosbahi K. Kim D. James R. Moore G.R. Kleanthous C. Protein Sci. 2006; 15: 620-627Crossref PubMed Scopus (23) Google Scholar). Colicin DNases also have the ability to form voltage-independent ion channels in planar lipid bilayers (5Mosbahi K. Kim C. Keeble A.H. Mobasheri H. Morel B. James R. Moore G.R. Lea E.J. Kleanthous C. Nat. Struct. Biol. 2002; 9: 476-484Crossref PubMed Scopus (44) Google Scholar). Considering that the E. coli IM is normally composed of 70–80% neutral phospholipids and 20–30% anionic phospholipids (31Kadner R.J. Curtis R. II I Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1996: 58-87Google Scholar), these experiments pointed to the possibility that colicin nucleases, which are positively charged domains, might interact directly with one or both of the membrane systems of E. coli en route to the cytoplasm. The present work set out to test this hypothesis. In addressing the importance of electrostatically driven protein-lipid interactions in the translocation of the E9 DNase domain, we used an E. coli strain depleted in anionic phospholipids. The strain HDL11 contains a single copy of the pgsA gene under the control of the lac operon and so is IPTG-inducible (18Kusters R. Kim W. de Kruijff B. J. Biol. Chem. 1991; 266: 8659-8662Abstract Full Text PDF PubMed Google Scholar). The product of the pgsA gene is responsible for the production of the major anionic phospholipid phosphatidylglycerol. In the absence of IPTG, HDL11 produces little phosphatidylglycerol (2%) or cardiolipin (1%) but does contain phosphatidic acid (6%) and so has a total content of anionic lipid of around 10% (18Kusters R. Kim W. de Kruijff B. J. Biol. Chem. 1991; 266: 8659-8662Abstract Full Text PDF PubMed Google Scholar). This is increased to ∼20% in the presence of 50 μm IPTG, with phosphatidylglycerol accounting for the majority (15%) as is the case in wild type K-12 strains (18Kusters R. Kim W. de Kruijff B. J. Biol. Chem. 1991; 266: 8659-8662Abstract Full Text PDF PubMed Google Scholar). The addition of concentrations of IPTG in excess of this does not increase the proportion of anionic phospholipid in the IM. We found that killing of strain HDL11 by an excess of colicin E9 (5 μg ml-1), where cell death was monitored by measurement of the optical density of the growing culture, was dependent on IPTG concentration (Fig. 1a). In the absence of IPTG, cell growth continued for several hours after the addition of the colicin, although cell death eventually ensues. In the presence of 50 μm IPTG, however, the rate of cell killing was restored to a level similar to that observed in wild type K-12 strains (data not shown). The addition of higher IPTG concentrations (100 or 500 μm) did not increase the level of cell killing further; with 20 or 30 μm IPTG, the level was intermediate between 0 and 50 μm (Fig. 1a). The data show that although HDL11 is not resistant to colicin E9, the kinetics of cell killing are strongly dependent on the level of anionic phospholipid expressed by the bacterium. The phospholipid composition of the IM bilayer and the inner leaflet of the OM is affected in the HDL11 strain. Consequently, the effect of IPTG concentration on colicin E9 toxicity could reflect changes at either surface. The outer leaflet of the OM is comprised predominantly of LPS and so is unlikely to be grossly affected in HDL11. Nevertheless, the phospholipid:LPS ratio of the OM would be expected to change, and this could affect early steps in colicin import involving receptor binding and translocation across the OM. To help address this issue, we analyzed the susceptibility of HDL11 to the group A pore-forming colicins E1 and A, which translocate across the OM by the same basic mechanism as nuclease colicins; both colicins parasitize BtuB as their primary receptor and then recruit OM translocators (TolC and OmpF, respectively) followed by translocation to the periplasm through binding of the Tol·Pal complex. We found that both pore-forming colicins killed HDL11 with identical killing profiles irrespective of whether IPTG was added to the culture (Fig. 1b), suggesting that the machinery for translocating colicins across the OM has remained largely unaltered. We conclude therefore that the effect of reduced anionic phospholipid content on colicin E9 toxicity most likely concerns effects at the IM. We also investigated other naturally occurring DNase type E colicins, E2, E7, and E8, which have been characterized extensively (32Pommer A.J. Kim U.C. Cooper A. Hemmings A.M. Moore G.R. James R. Kleanthous C. J. Biol. Chem. 1999; 274: 27153-27160Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 33van den Bremer E.T. Kim A.H. Jiskoot W. Spelbrink R.E. Maier C.S. van Hoek A. Visser A.J. James R. Moore G.R. Kleanthous C. Heck A.J. Protein Sci. 2004; 13: 1391-1401Crossref PubMed Scopus (13) Google Scholar). DNase colicins are 60-kDa toxins that share a high degree of sequence identity in their receptor-binding and translocation domains (>90%) but are less conserved in their respective 15-kDa DNases (∼65%), and although all are basic domains (pI > 9.6), they differ in the number and type of charged residues. In particular, the net positive charge for all four DNase domains varies considerably, with E7, E2, E8, and E9 having +13, +11, +9, and +7 charges, respectively. We found that in contrast to colicin E9, where killing of E. coli HDL11 was dependent on the IPTG concentration, colicins E7 (Fig. 1c) or E2 (Fig. 1d) were equally cytotoxic against HDL11 regardless of whether IPTG was added to the culture (only data in the absence of IPTG are shown in Fig. 1d). Thus, for the most positively charged variants reducing the proportion of negatively charged lipid does not retard the level of cell killing. With colicin E8 (+9) (data not show) the absence of IPTG had some effect on cell killing, but not to the extent observed with colicin E9 (+7) (Fig. 1c). DNase E colicins are essentially identical in the sequences of their receptor-binding and translocation domains, and so these are unlikely to explain the differing behavior of these toxins on HDL11. Two effects could reasonably account for the influence of colicin DNase charge and inducing agent on cell killing: (i) The endonucleolytic activities and hence cytotoxicities of the most positively charged variants (E2 and E7) are significantly greater than those of E8 and E9. This can be discounted because a comparison of the enzymatic activities of all DNase colicins shows no such trend. Indeed, colicin E8 has the greatest relative activity in vitro in both plasmid-nicking and spectrophotometric assays, with E2, E7, and E9 having approximately equivalent activities (33van den Bremer E.T. Kim A.H. Jiskoot W. Spelbrink R.E. Maier C.S. van Hoek A. Visser A.J. James R. Moore G.R. Kleanthous C. Heck A.J. Protein Sci. 2004; 13: 1391-1401Crossref PubMed Scopus (13) Google Scholar). (ii) The differing net positive charge of the DNases affects their ability to associate with the IM of HDL11. If this explanation is correct, then we rationalized it should be possible to engineer enhanced or diminished cell killing activity merely by increasing or decreasing, respectively, the positive charge on a single colicin nuclease domain. This would also discount the possibility that the different levels of activity against HDL11 was due to subtle structural differences between the enzymes of the DNase colicin family and so unrelated to the amount of positive charge. To test this hypothesis we engineered charge variants in the DNase of colicin E9, both increasing and decreasing the net positive charge. Positions outside of the enzyme active site were chosen that are not involved in catalysis or DNA binding (34Mate M.J. Kim C. J. Biol. Chem. 2004; 279: 34763-34769Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Single and double Lys-to-Glu substitutions (at Lys21 and Lys45; numbering for the isolated domain) were engineered to reduce the amount of positive charge to +5 and +3, respectively. Both mutants showed reduced killing against HDL11 relative to the wild type colicin E9, with the double mutant more strongly impaired (Fig. 1d). In contrast, the introduction of additional positive charges into the DNase domain of colicin E9 (S77K/S78R and S77K/S78R/S80K) showed enhanced cell killing of HDL11 relative to wild type colicin E9 (Fig. 1d). We tested the enzymatic activities of all the engineered charge variants and found no correlation with their biological activity; indeed, both sets of mutants had slightly reduced plasmid DNA nicking activities relative to wild type colicin E9 (data not shown). Our data demonstrate that the cell killing efficiency of a colicin DNase against E. coli HDL11 with reduced anionic phospholipid content can be enhanced or reduced merely by changing the number of net positive charges and that this is not due to differences in enzymatic activity between the colicin DNases. To ascertain whether there is a quantitative relationship between cell killing ability and colicin DNase charge, we plotted all of the HDL11 IPTG cell killing data (quantified as the change in A600 following the addition of colicin) as a function of the positive charge of the domain. From the resulting plot it is clear that there is a strong correlation between the net positive charge on a colicin DNase domain and its cytotoxicity activity against HDL11 (Fig. 2). We also looked for systematic variations in toxicity when HDL11 was induced with 100 μm IPTG and compared this to a wild type strain of E. coli K-12 (SD12). In both cases a weak correlation with enzyme charge was apparent (data not shown) but was less clear-cut; the most-to-least positively charged variants displayed only small differences in optical density (<0.3) compared with HDL11-IPTG. Hence, the correlation between enzyme charge and cytotoxicity, at least as measured by changes in culture optical density, is masked when the IM carries wild type negative charge and only becomes readily apparent in E. coli that is depleted of anionic phospholipids. Electrostatic Interactions Gate Entry of DNase Colicins into Wild Type E. coli Cells—To probe further the involvement of electrostatic interactions in colicin entry, particularly in wild type strains, and to provide quantitative date on cell entry kinetics, we resorted to trypsin-protection assays of colicin toxicity. Trypsin rapidly degrades colicins, rendering them inactive, and has been used extensively to investigate colicin entry kinetics (35Braun V. Kim J. Hantke K. Schaller K. J. Bacteriol. 1980; 142: 162-168Crossref PubMed Google Scholar, 36Benedetti H. Kim R. Lazdunski C. Letellier L. EMBO J. 1992; 11: 441-447Crossref PubMed Scopus (98) Google Scholar). Trypsinolysis of colicin-treated cells was used by Benedetti et al. (36Benedetti H. Kim R. Lazdunski C. Letellier L. EMBO J. 1992; 11: 441-447Crossref PubMed Scopus (98) Google Scholar) to show that the pore-forming toxin colicin A is unfolded and exposed to the extracellular environment even when its cytotoxic domain is depolarizing the IM. Similar experiments have been reported recently by Duché (37Duché D. J. Bacteriol. 2007; 189: 4217-4222Crossref PubMed Scopus (23) Google Scholar) for the DNase colicin E2, demonstrating that the toxin remains attached to its OM receptor while contacting the Tol proteins in the periplasm. Colicins must span large distances (100–200 Å) to simultaneously contact one or more proteins in the OM and periplasm, and this likely explains why colicins such as E3 and Ia have elongated hairpin structures with long coiled-coil regions connecting their cytotoxic and translocation domains (38Wiener M. Kim D. Ghosh P. Stroud R.M. Nature. 1997; 385: 461-464Crossref PubMed Scopus (223) Google Scholar, 39Soelaiman S. Kim K. Wu N. Li C. Shoham M. Mol. Cell. 2001; 8: 1053-1062Abstract Full Text Full Text PDF PubMed Scopus (

Referência(s)