JB
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
JB.00787-06v1
188/22/7957    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Banerjee, J.
Right arrow Articles by Banerjee, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Banerjee, J.
Right arrow Articles by Banerjee, N.

 Previous Article  |  Next Article 

Journal of Bacteriology, November 2006, p. 7957-7962, Vol. 188, No. 22
0021-9193/06/$08.00+0     doi:10.1128/JB.00787-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The Cytotoxic Fimbrial Structural Subunit of Xenorhabdus nematophila Is a Pore-Forming Toxin{triangledown}

Jyotirmoy Banerjee,3,{ddagger} Jitendra Singh,1,2,{ddagger} Mohan Chandra Joshi,1,2 Shubhendu Ghosh,3 and Nirupama Banerjee1*

International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India,1 Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India,2 Department of Biophysics, University of Delhi, New Delhi 110021, India3

Received 1 June 2006/ Accepted 16 August 2006


   ABSTRACT
Top
 Abstract
Text
 References
 
We have purified a fimbrial shaft protein (MrxA) of Xenorhabdus nematophila. The soluble monomeric protein lysed larval hemocytes of Helicoverpa armigera. Osmotic protection of the cells with polyethylene glycol suggested that the 17-kDa MrxA subunit makes pores in the target cell membrane. The internal diameter of the pores was estimated to be >2.9 nm. Electron microscopy confirmed the formation of pores by the fimbrial subunit. MrxA protein oligomerized in the presence of liposomes. Electrophysiological studies demonstrated that MrxA formed large, voltage-gated passive-diffusion channels in lipid bilayers.


   TEXT
Top
 Abstract
 Text
References
 
Xenorhabdus nematophila is an insect-pathogenic bacterium residing in the intestinal vesicle of a soil nematode of the genus Steinernema as a symbiont (1, 10, 11). X. nematophila is adapted to two diverse environmental niches, the nematode gut and the insect larval body, in which it produces potent toxins to kill its larval prey (6, 7, 24, 29).

We are studying the secretome of X. nematophila to identify effector molecules produced by the latter for interacting with its larval and nematode hosts (15). In gram-negative bacteria, fimbriae form rigid, hair-like fibers on the cell surface and are known to play a key role in recognition and adherence of the pathogen to the host cell (12, 27, 35). Earlier investigations to identify novel insecticidal molecules and effectors mediating the pathogenicity of this insect pathogen (15) led to the serendipitous discovery of toxicity in the structural subunit (MrxA) of type 1 fimbriae of X. nematophila (16). In view of the urgent need to identify and develop novel insecticidal molecules for eradication of insect pests, we conducted this study to gain further insight into the mechanism of toxicity of pilin subunit MrxA of X. nematophila.

Cytolysis of insect hemocytes by 17-kDa MrxA suggested that membranes could be the primary targets of its action. Since no protease or lipase activity was detected in MrxA, we examined the pore-forming potential of the latter in hemocyte cultures. Many cytotoxic bacterial proteins are known to act through formation of pores in host cell membrane (3, 4, 5), resulting in a multitude of cellular responses, which may be relevant to the pathogenicity of the bacterium. The insecticidal Cry toxins produced by Bacillus thuringiensis are well-known examples of pore-forming toxins of invertebrate cells (13, 22). In Escherichia coli, a closely related species, three different pore-forming cytolysins, {alpha}-hemolysin, enterohemorrhagic E. coli hemolysin, and cytolysin A (18, 26, 32), have been identified so far. Pore-forming toxic proteins facilitate entry of the pathogen and/or damage the host cell for their own ends. In addition to the orally toxic proteins (7), X. nematophila is reported to produce a 10-kDa cytotoxic, cation-selective channel-forming protein in the culture medium (30). To understand the mode of action of cytotoxic fimbrial subunit MrxA of X. nematophila, we have investigated the pore-forming activity of the protein. Our investigations for the first time revealed a hitherto unknown property of the structural subunit of type 1 pilin of X. nematophila.

Bacterial growth and purification of monomeric 17-kDa MrxA subunit. X. nematophila 19061 was grown on nutrient agar plates (154 cm2) at 29 ± 1°C for 48 h. A partially purified protein (Fig. 1B, lanes 1 and 2) was obtained after ammonium sulfate precipitation and sucrose density gradient centrifugation (25, 17). The protein mixture was subjected to gel filtration through a Superose 12 column (bed volume, 24 ml; Pharmacia) in the fast protein liquid chromatography system. The column was equilibrated with 50 mM sodium phosphate buffer, pH 8, containing 150 mM NaCl. The proteins were loaded onto and eluted from the column in the same buffer. The elution profile of the proteins is shown in Fig. 1A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the column fractions showed that peak 1, eluting in the void volume, contained the 17-kDa MrxA protein as high-molecular-mass oligomers (>2,000 kDa) along with a 60-kDa protein (data not shown). Minor peaks 2 and 3 also contained smaller amounts of the 17-kDa protein in tetrameric and dimeric forms, respectively (data not shown). Major peak 4, eluting between 37 and 41 min, contained pure monomeric MrxA protein (Fig. 1B, lanes 3 and 4). The fractions eluting between 37 and 40 min were pooled and dialyzed against 10 mM Tris-HCl buffer, pH 8, overnight and used in all of the subsequent studies. Resolution of 17-kDa MrxA into multiple peaks by the sizing column indicated that the protein preparation obtained after density gradient centrifugation was a mixture of various oligomeric forms, including monomers. The purity and identity of the monomeric protein were confirmed by silver staining (Fig. 1B, lane 4) and peptide mass fingerprinting with an Agilent 1100 series2DnanoLC MS machine (data not shown). The structural integrity of the monomeric protein was ascertained by circular-dichroism spectroscopy (Fig. 1C). Thus, isolation of structurally stable and biologically active monomeric pilin subunit MrxA demonstrates the distinctive characteristics of type I fimbriae of X. nematophila. To date, no fimbrial subunit has been reported to exist independently in a stable form. It is necessary to mention here that monomers and oligomers of various sizes were observed routinely in the culture supernatant of X. nematophila (data not shown), reflecting weaker interactions between the protomers or inadequate capping of the pilin fiber, resulting in random breaks during growth.


Figure 1
View larger version (31K):
[in this window]
[in a new window]
  		FIG. 1. Purification and characterization of monomeric pilin structural subunit MrxA. (A) Elution profile of the protein from a Superose 12 column. Symbols: {blacksquare}, E. coli fimbriae; •, X. nematophila fimbrial protein. (B) SDS-PAGE profile of 17-kDa MrxA at different stages of purification. Samples were heated with Laemmli's sample dye containing 2% SDS and 200 mM dithiothreitol at 95°C for 5 min and loaded onto the gels. The gel was stained with Coomassie blue. Lane 1, crude pilin protein; lane 2, protein after sucrose density gradient centrifugation; lane 3, 17-kDa MrxA obtained in peak 4 from a Superose 12 column; lane 4, 2 µg of the protein used in lane 3 heated with Laemmli's sample dye for 5 min at 95°C and silver stained; lane 5, purified pilin protein from E. coli K-12; lanes M, molecular mass markers. The values on the right are molecular sizes in kilodaltons. (C) Far UV spectrum of the purified protein. Molar ellipticity is represented by {theta} in degree · square centimeters per decimole.

 
The homologous FimA protein was purified from E. coli K-12 (19) (Fig. 1B, lane 5). However, unlike that from X. nematophila, most of the protein was obtained as large oligomers from the Superose column, showing inherent dissimilarities between subunits constituting the fimbrial shaft of E. coli and X. nematophila. FimA was used as a control in electrophysiology and cytotoxicity assays.

MrxA forms pores in insect hemocytes. Hemocytes from fifth-instar Helicoverpa armigera larvae were isolated and cultured in 96-well tissue culture plates in 100 µl of Grace's serum-free insect cell culture medium (16). The cells were incubated with purified protein at 27°C for 1 to 2 h. The lactate dehydrogenase (LDH) released in the supernatant was measured as an indicator of cytolysis with a Cyto-Tox (Promega) kit. To examine if cytolysis occurred because of pore formation by MrxA, osmotic protection of the cells by polyethylene glycol (PEG) was performed in the presence of 10 µg/well protein, which caused ~50% lysis of the cells in 2 h, and 30 mM PEG with molecular sizes varying from PEG 1000 to PEG 20000. In a duplicate set of experiments, the osmotically protected cells were rinsed and transferred to phosphate-buffered saline and cytolysis was determined. The experiment was repeated at least three times, and the values represent the mean ± the standard deviation of triplicate samples from one representative experiment. Percent protection was calculated on the basis of LDH released by the cells in the absence of any additive as 100% and in the presence of protein alone as 0%.

The purified MrxA protein caused swelling of larval hemocytes, followed by cytolysis in a dose-dependent manner (Fig. 2A). Galactose, glucuronic acid, gluconic acid, and xylitol protected the cells from the cytolytic action of MrxA (data not shown). Heat inactivation of the protein at 60°C for 15 min abolished its toxicity. It is to be noted that the larger aggregates and oligomers of the 17-kDa protein in peak 1 were not toxic to the cells from 1 to 20 µg/well; possible reasons for inactivity could be that (i) the aggregates are denatured protein and hence inactive and (ii) they are larger fragments (oligomers) of the pilin shaft that are unable to insert themselves into the membrane efficiently because of steric constraints. Moreover, it is clear that the structural organization of MrxA oligomers present in peak 1 is fundamentally different from that of those produced in the presence of lipid membranes (shown later). Homologous protein FimA from E. coli K-12 showed no activity on larval hemocytes (Fig. 2A). Since FimA was present predominantly as large oligomeric fragments, the exact reason for its inactivity remains unresolved at this stage.


Figure 2
View larger version (11K):
[in this window]
[in a new window]
  		FIG. 2. Effect of MrxA on larval hemocytes and their protection by osmotic protectants. (A) Freshly prepared hemocytes from fourth- to fifth-instar larvae were incubated with 1 to 20 µg/well purified 17-kDa protein; peak 1 protein at 20 µg/well; heat-inactivated purified protein at 10 µg/well; and E. coli pilin protein at 10 µg/well. After 2 h, the supernatant was removed carefully for LDH estimation. The cells were lysed with Triton X-100, and total LDH was determined; degree of lysis was determined as a percentage of fully lysed cells. (B) Hemocytes were incubated with 10 µg/well protein and 30 mM PEGs with different molecular sizes for 1 to 2 h, and the extent of cellular lysis was determined by measuring the amount of LDH released. Results are presented as a percentage of the control (cells without any additive). The values are averages of triplicate wells with error bars showing standard deviations.

 
The cytolytic activity of MrxA was reduced substantially in the presence of PEG 6000 and above (Fig. 2), while PEG 4000 and below provided very little or no protection. Increasing the protein concentration to 20 µg/well reduced the protective effect of PEG 6000 only by 12%. No protection was observed in the presence of PEG 1000 or smaller sugars such as glucose with molecular diameters of 1.0 nm and less. When the cells protected by PEG 6000 were washed and resuspended in buffer, lysis occurred instantly, indicating that the PEG did not influence insertion of the protein into the membrane. Osmotic protection of H. armigera hemocytes by PEG demonstrates that the toxicity of MrxA is mediated by colloid-osmotic lysis forming pores in the host cell membranes. Based on the protection of cells by PEG, the pore size was estimated as >2.9 nm, denoting the hydrodynamic radii of PEG 6000. Ion channels formed by different bacterial toxins such as CAMP factor from Streptococcus agalactiae (1.6 to 15 nm) (20), hydralysin from cnidaria (1.2 nm) (33), ClyA cytolysin from E. coli (2.6 to 5 nm) (23, 36), and {alpha}-xenorhabdolysin from X. nematophila (2 to 3 nm) (30), etc., also fall in this range.

MrxA forms oligomers in liposomes. Oligomerization of the protein in the membrane was examined by chemical cross-linking with glutaraldehyde. The liposomes were prepared from a 1% solution of cholesterol and diphytanoyl phosphatidylcholine (DPhPC) (1:6) in n-decane. The lipids were evaporated to dryness under a stream of nitrogen, and buffer (500 mM KCl, 10 mM HEPES, 5 mM MgCl2, pH 7.4) was added to the lipid mixture. The mixture was kept in a sonic bath for 30 min. Purified protein (8 to 10 µg) was added to 20 µl of liposomes, and the mixture was incubated at 37°C for 30 min. The protein-liposome complex was incubated with 2.5 mM glutaraldehyde for 30 min, heated with SDS loading buffer at 95°C for 5 min, and resolved by SDS-PAGE.

MrxA formed SDS-resistant oligomers in the presence of liposomes. A substantial part of the protein remained at the junction of stacking and a resolving gel (Fig. 3, lanes 3 and 4), even in the absence of glutaraldehyde cross-linking (Fig. 3, lane 4). Cross-linking of the protein in the absence of liposomes produced no oligomers (Fig. 3, lane 2). The pore-forming toxins aerolysin of Aeromonas sobria (8) and epsilon toxin of Clostridium perfringens (28) were also shown to produce SDS-resistant oligomers in liposomes. These results suggested that the presence of lipids triggers a conformational change in the monomer, exposing the hydrophobic domains for insertion in the nonpolar environment of the membrane. Such conformational changes (14, 21), followed by oligomerization on the surface of the target membrane, have been reported in many pore-forming, water-soluble proteins (9, 34).


Figure 3
View larger version (115K):
[in this window]
[in a new window]
  		FIG. 3. Oligomerization of 17-kDa MrxA in liposomes. Purified protein was incubated with 20 µl of liposomes prepared from cholesterol-DPhPC (1:6) at 37°C for 30 min. The liposome-protein complex was cross-linked with 2.5 mM glutaraldehyde for 30 min, and the samples were denatured by heating at 95°C for 5 min before loading on SDS-PAGE. Lane 1, liposomes alone; lane 2, 17-kDa protein cross-linked with glutaraldehyde; lane 3, lipid-protein complex cross-linked with glutaraldehyde; lane 4, lipid-protein complex without cross-linking with glutaraldehyde; lane M, molecular size markers (sizes on the right are in kilodaltons).

 
Electron microscopy of pores formed by MrxA. Liposomes were prepared as described above and resuspended in 25 µl of reconstitution buffer. Ten microliters of purified MrxA (200 µg/ml) was added to the liposomes, and the mixture was incubated at room temperature for 3 to 4 h. The samples were adsorbed on carbon-coated copper grids and stained with a 1% solution of sodium phosphotungstate and visualized in a Morgagni 268D microscope at 100 kV. A negatively stained preparation of the membrane complex showed that MrxA formed discrete pores in the lipid membrane (Fig. 4B). The pores were round and regular in shape. Most of the pores ranged between 3 and 5 nm in internal diameter, which is in agreement with osmotic-protection studies. A smaller number appeared to range between 8 and 10 nm in diameter, suggesting that larger assemblies of subunits are also possible. Similar variations in size were reported for pores formed by the hemolytic protein HlyE in the artificial lipid membranes (36). The size variation could be due to differences in the lipid composition of the membranes in liposomes and the target cells, allowing variable numbers of subunits to assemble in the pore. Toxin proteins producing pores of different size in different cell types have been reported earlier (30).


Figure 4
View larger version (118K):
[in this window]
[in a new window]
  		FIG. 4. Electron micrographs of pores formed by MrxA in lipid vesicles. The protein-liposome complex was incubated at room temperature for 3 to 4 h, negatively stained with 1% Na phosphotungstate for 1 min, and visualized in a Morgagni 268D microscope run at 100 kV. Panels: A, liposomes without protein; B, liposomes incubated with purified protein; C, purified MrxA protein alone. The scale bars represent 60 nm.

 
Electrophysiological properties of MrxA channels in lipid bilayers. The MrxA protein was inserted into lipid bilayers as described earlier (31). Briefly, the apparatus used consisted of a polystyrene cuvette (Warner Instrument Corp.) divided into two compartments by a thin divider with a circular aperture of 150 µm. The two compartments were filled with buffer (500 mM KCl or NaCl, 5 mM MgCl2, 10 mM HEPES, pH 7.4) connected to an integrating patch amplifier (Axopatch 200A; Axon Instruments) through a matched pair of Ag-AgCl electrodes. The membrane was formed with a mixture of cholesterol and DPhPC in n-decane (1:6, wt/wt), and the protein was added gradually to the buffer in the cis chamber. The Axopatch 200A was connected to an IBM computer through a Digidata 1322A interface (Axon Instruments). Single-channel recordings were obtained at clamping potentials of –120 mV to +120 mV. Steady-state current was calculated from the single-channel current data with the software Clampfit (pClamp 9.0; Axon Instruments). The probability of channel opening (Po) was calculated as described earlier (2). Continuous current traces of 1 min for each clamping potential were taken for calculation of Po. Once a single-channel current was obtained, 50 µl of diluted (1:100) polyclonal antibodies against the 17-kDa protein or preimmune serum was added gradually to the cis chamber.

The MrxA protein formed passive diffusion channels in the lipid bilayer membrane. The membrane conductance increased after 30 min of addition of protein. No membrane current was observed up to 350 ng/ml protein (Fig. 5A); at 400 ng/ml and 60 mV, the specific membrane conductance increased several orders of magnitude. The current was symmetrical with respect to the positive or negative polarity of the electrical field. The channel showed gating over a voltage range of ±60 mV to ±120 mV (Fig. 5B, C, D, and E). Ionic conductance was similar in the presence of 500 mM KCl, NaCl, or sodium citrate (data not shown), indicating the nonselective nature of the pore. The pores formed in lipid bilayers showed a long life span of 40 to 45 min. Oligomeric MrxA from peak 1 showed no channel conductance, matching its low toxicity on the cells (Fig. 5H). All of the experiments were repeated at least three times, and the data are representative of one of them. The steady-state current-voltage curve (Fig. 5M) shows a linear increase in channel current with applied voltage. Similarly, Po was also dependent on voltage and was highest at ±80 mV (Fig. 5N). The high channel conductance of the pore formed by MrxA (between 0.33 nS and 0.99 nS) indicates a large pore size (Fig. 5O), supporting the earlier observations of oligomerization and formation of 3- to 10-nm pores in liposomes and cells. Heating the protein to 60°C abolished its channel-forming activity completely (data not shown). Antibodies against the pilin subunit caused a sharp decrease in the channel current, while preimmune serum had no effect on channel conductance (Fig. 5F and G). Addition of antibodies had no effect on membrane integrity. Similar disruptions in current were observed consistently at all applied potentials (Fig. 5O). Homologous pilin subunit FimA of E. coli showed no channel conductance when incorporated into the lipid membrane under the above-described conditions (data not shown), indicating that the ion-conducting property is specific for the X. nematophila pilin subunit. The ability of recombinant MrxA to produce channels in lipid bilayer membranes with similar electrophysiological properties (Fig. 5I, J, K, and L) strengthens the claim that pore formation is indeed due to pilin structural subunit MrxA and not to any contaminating protein. The properties of the pores formed by MrxA of X. nematophila in lipid bilayers are in good agreement with those formed by other pore-forming bacterial toxins like aerolysin (8), the ClyA protein of E. coli (23), and the CAMP factor of S. agalactiae (20).


Figure 5
View larger version (33K):
[in this window]
[in a new window]
  		FIG. 5. Electrophysiology of pores formed by MrxA in lipid bilayers. Continuous single-channel current traces were recorded at 25°C in 500 mM KCl-10 mM HEPES-5 mM MgCl2, pH 7.4. Shown are conductances at ±50 mV (A); 30 min after protein addition at +60 mV (B), –60 mV (C), +120 mV (D), and –120 mV (E); at +80 mV after addition of anti-MrxA antibody (F); after addition of preimmune serum at +80 mV (G); after addition of oligomeric 17-kDa protein from peak 1 at ±80 mV (H); after addition of recombinant MrxA protein at +60 mV (I), –60 mV (J), +80 mV, (K), and –80 mV (L); the steady-state current-voltage relationship of the pore in symmetrical bathing buffer (M); the dependence of Po on voltage (N); and the conductance profiles of the channels formed by MrxA protein ({blacksquare}) and antibody-treated protein ({square}) (O). The data shown were taken from continuous current recordings of 1 min at each holding potential. Values are means ± standard deviations of three independent experiments.

 
In conclusion, our results for the first time demonstrate ion-conducting channels formed by a fimbrial subunit of a gram-negative bacterium. Its ability to insert itself, oligomerize, and modify membrane permeability correlates directly with its cytotoxic effect on larval hemocytes. Association of toxicity with the structural subunit forming the fimbrial shaft, an extracellular organelle normally required for recognition of pathogens and attachment to the host, appears to be an ingenious way, indeed, to ensure transport and targeting of the toxin to the host cell.


   ACKNOWLEDGMENTS
 
We thank CSIR, Government of India, for financial assistance to J.S., M.J., and J.B.

Thanks to G. K. Gupta for providing H. armigera larvae for hemocyte isolation.


   FOOTNOTES
 
* Corresponding author. Mailing address: International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. Phone: 91-11-26181242. Fax: 91-11-26162316. E-mail: nirupama{at}icgeb.res.in. Back

{triangledown} Published ahead of print on 1 September 2006. Back

{ddagger} J.B. and J.S. contributed equally to this study. Back


   REFERENCES
Top
 Abstract
 Text
 References
 

  1. Akhurst, R. J., and G. B. Dunphy. 1993. Symbiotically associated entomopathogenic bacteria, nematodes and their insect hosts, p. 1-23. In N. Beckage, S. Thompson, and B. Federici (ed.), Parasites and pathogens of insects. Academic Press, Inc., New York, N.Y.
  2. Aldrich, R. W., D. P. Corey, and C. F. Stevens. 1983. A reinterpretation of mammalian sodium channel gating based on single channel recording. Nature 306:436-441.[CrossRef][Medline]
  3. Alouf, J. E. 2001. Pore-forming bacterial protein toxins: an overview. Curr. Top. Microbiol. Immunol. 257:1-14.[Medline]
  4. Ballard, J., J. Crabtree, B. A. Roe, and R. K. Tweten. 1995. The primary structure of Clostridium septicum alpha-toxin exhibits similarity with that of Aeromonas hydrophila aerolysin. Infect. Immun. 63:340-344.[Abstract]
  5. Bhakdi, S., and J. Tranum-Jensen. 1985. Complement activation and attack on autologous cell membranes induced by streptolysin-O. Infect. Immun. 48:713-719.[Abstract/Free Full Text]
  6. Brillard, J., C. Ribeiro, N. Boemare, M. Brehelin, and A. Givaudan. 2001. Two distinct hemolytic activities in Xenorhabdus nematophila are active against immunocompetent insect cells. Appl. Environ. Microbiol. 67:2515-2525.[Abstract/Free Full Text]
  7. Brown, S. E., A. T. Cao, E. R. Hines, and R. J. Akhurst. 2004. A novel secreted protein toxin from the insect-pathogenic bacterium Xenorhabdus nematophila. J. Biol. Chem. 279:14595-14601.[Abstract/Free Full Text]
  8. Chakraborty, T., A. Schmid, S. Notermans, and R. Benz. 1990. Aerolysin of Aeromonas sobria: evidence for formation of ion-permeable channels and comparison with alpha-toxin of Staphylococcus aureus. Infect. Immun. 58:2127-2132.[Abstract/Free Full Text]
  9. Czajkowsky, D. M., E. M. Hotze, Z. Shao, and R. K. Tweten. 2004. Vertical collapse of a cytolysin prepore moves its transmembrane beta-hairpins to the membrane. EMBO J. 23:3206-3215.[CrossRef][Medline]
  10. Forst, S., and K. Nealson. 1996. Molecular biology of the symbiotic-pathogenic bacteria Xenorhabdus spp. and Photorhabdus spp. Microbiol. Rev. 60:21-43.[Free Full Text]
  11. Forst, S., B. Dowds, N. Boemare, and E. Stackebrandt. 1997. Xenorhabdus and Photorhabdus spp.: bugs that kill bugs. Annu. Rev. Microbiol. 51:47-72.[CrossRef][Medline]
  12. Gaastra, W., and E. K. DeGraaf. 1992. Host specific fimbrial adhesin of non-invasive enterotoxigenic Escherichia coli strains. Microbiol. Rev. 46:129-161.
  13. Gouaux, E. 1997. Channel-forming toxins: tales of transformation. Curr. Opin. Struct. Biol. 7:566-573.[CrossRef][Medline]
  14. Hughson, F. M. 1997. Penetrating insights into pore formation. Nat. Struct. Biol. 4:89-92.[CrossRef][Medline]
  15. Khandelwal, P., and N. Banerjee-Bhatnagar. 2003. Insecticidal activity associated with the outer membrane vesicles of Xenorhabdus nematophilus. Appl. Environ. Microbiol. 69:2032-2037.[Abstract/Free Full Text]
  16. Khandelwal, P., D. Choudhury, A. Birah, M. K. Reddy, G. P. Gupta, and N. Banerjee. 2004. An insecticidal pilin subunit from insect pathogenic bacterium Xenorhabdus nematophila. J. Bacteriol. 186:6465-6476.[Abstract/Free Full Text]
  17. Khandelwal, P., D. Choudhury, R. Bhatnagar, and N. Banerjee. 2004. Characterization of a cytotoxic pilin subunit of Xenorhabdus nematophila. Biochem. Biophys. Res. Commun. 314:943-949.[CrossRef][Medline]
  18. Konig, B., A. Ludwig, W. Goebel, and W. Konig. 1994. Pore formation by the Escherichia coli alpha-hemolysin: role for mediator release from human inflammatory cells. Infect. Immun. 62:4611-4617.[Abstract/Free Full Text]
  19. Korhonen, T. K., E. L. Nurmiaho, H. Ranta, and C. S. Eden. 1980. New method for isolation of immunologically pure pili from Escherichia coli. Infect. Immun. 27:569-575.[Abstract/Free Full Text]
  20. Lang, S., and M. Palmer. 2003. Characterization of Streptococcus agalactiae CAMP factor as a pore-forming toxin. J. Biol. Chem. 278:38167-38173.[Abstract/Free Full Text]
  21. Lesieur, C., B. Vecsey-Semjen, L. Abrami, M. Fivaz, and F. Gisou van der Goot. 1997. Structure of the Aeromonas toxin proaerolysin in its water-soluble and membrane-channel states. Mol. Membr. Biol. 14:45-64.[Medline]
  22. Li, J. D., J. Carroll, and D. J. Ellar. 1991. Crystal structure of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5 Å resolution. Nature 1353:815-821.
  23. Ludwig, A., S. Bauer, R. Benz, B. Bergmann, and W. Goebel. 1999. Analysis of the SlyA-controlled expression, subcellular localization and pore-forming activity of a 34 kDa haemolysin (ClyA) from Escherichia coli K-12. Mol. Microbiol. 31:557-567.[CrossRef][Medline]
  24. Morgan, J. A., M. Sergeant, D. Ellis, M. Ousley, and P. Jarrett. 2001. Sequence analysis of insecticidal genes from Xenorhabdus nematophilus PMFI296. Appl. Environ. Microbiol. 67:2062-2069.[Abstract/Free Full Text]
  25. Moureaux, N., T. Karjalainen, A. Givaudan, P. Bourlioux, and N. Boemare. 1995. Biochemical characterization and agglutinating properties of Xenorhabdus nematophilus F1 fimbriae. Appl. Environ. Microbiol. 61:2707-2712.[Abstract]
  26. Oscarsson, J., Y. Mizunoe, L. Li, X. H. Lai, A. Wieslander, and B. E. Uhlin. 1999. Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli. Mol. Microbiol. 32:1226-1238.[CrossRef][Medline]
  27. Pearce, W. A., and T. M. Buchanan. 1978. Attachment role of gonococcal pili. Optimum conditions and quantification of adherence of isolated pili to human cells in vitro. J. Clin. Investig. 61:931-943.[Medline]
  28. Petit, L., M. Gibert, D. Gillet, C. Laurent-Winter, P. Boquet, and M. R. Popoff. 1997. Clostridium perfringens epsilon toxin acts on MDMK cells by forming a large membrane complex. J. Bacteriol. 179:6480-6487.[Abstract/Free Full Text]
  29. Ribeiro, C., B. Duvic, P. Oliveira, A. Givaudan, F. Palha, N. Simoes, and M. Brehelin. 1999. Insect immunity—effects of factors produced by a nematobacterial complex on immunocompetent cells. J. Insect Physiol. 45:677-685.[CrossRef][Medline]
  30. Ribeiro, C., M. Vignes, and M. Brehelin. 2003. Xenorhabdus nematophila (Enterobacteriaceae) secretes a cation-selective calcium-independent porin which causes vacuolation of the rough endoplasmic reticulum and cell lysis. J. Biol. Chem. 278:3030-3039.[Abstract/Free Full Text]
  31. Roos, N., R. Benz, and D. Brdiczka. 1982. Identification and characterization of the pore-forming protein in the outer membrane of rat liver mitochondria. Biochim. Biophys. Acta 686:204-214.[Medline]
  32. Schmidt, H., E. Maier, H. Karch, and R. Benz. 1996. Pore-forming properties of the plasmid-encoded hemolysin of enterohemorrhagic Escherichia coli O157:H7. Eur. J. Biochem. 241:594-601.[Medline]
  33. Sher, D., F. Fishman, M. Zhang, M. Lebendiker, A. Gaathon, J. Mancheño, and E. Zlotkin. 2005. Hydralysins, a new category of ß-pore-forming toxins in Cnidaria. J. Biol. Chem. 280:22847-22855.[Abstract/Free Full Text]
  34. Tilley, S. J., E. V. Orlova, R. J. Gilbert, P. W. Andrew, and H. R. Saibil. 2005. Structural basis of pore formation by the bacterial toxin pneumolysin. Cell 121:247-256.[CrossRef][Medline]
  35. Tinker, J. K., L. S. Hancox, and S. Clegg. 2001. FimW is a negative regulator affecting type 1 fimbrial expression in Salmonella enterica serovar Typhimurium. J. Bacteriol. 183:435-442.[Abstract/Free Full Text]
  36. Wallace, A. J., T. J. Stillman, A. Atkins, S. J. Jamieson, P. A. Bullough, J. Green, and P. J. Artymiuk. 2000. E. coli hemolysin E (HlyE, ClyA, SheA): X-ray crystal structure of the toxin and observation of membrane pores by electron microscopy. Cell 100:265-276.[CrossRef][Medline]

Journal of Bacteriology, November 2006, p. 7957-7962, Vol. 188, No. 22
0021-9193/06/$08.00+0     doi:10.1128/JB.00787-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
JB.00787-06v1
188/22/7957    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Banerjee, J.
Right arrow Articles by Banerjee, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Banerjee, J.
Right arrow Articles by Banerjee, N.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Appl. Environ. Microbiol. Infect. Immun. Eukaryot. Cell
Mol. Cell. Biol. J. Virol. Microbiol. Mol. Biol. Rev.
ALL ASM JOURNALS