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Chapter 45 : Identification of Translocated Substrates of the Legionella pneumophila Dot/Icm System without the use of Eukaryotic Host Cells
Authors:
Ralph R. Isberg1,
Matthias Machner2
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Affiliations:
1: Howard Hughes Medical Institute and Dept. of Molecular Biology and Microbiology, Tufts University School of Medicine, 150 Harrison Ave., Boston, MA 02111;
2: Howard Hughes Medical Institute and Dept. of Molecular Biology and Microbiology, Tufts University School of Medicine, 150 Harrison Ave., Boston, MA 02111
Content Type: Monograph
Publication Year:
2006
Category: Bacterial Pathogenesis
Book DOI:
10.1128/9781555815660
Chapter DOI:
10.1128/9781555815660.ch45
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Identification of Translocated Substrates of the Legionella pneumophila Dot/Icm System without the use of Eukaryotic Host Cells, Page 1 of 2
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Abstract:
This chapter describes alternate approaches that take advantage of special properties of Legionella pneumophila strains that allowed the formulation of simple screens using toothpicks and petri dishes containing bacteriological medium. Developing readouts in the microorganism that allow screens on solid medium has allowed a number of translocated substrates to be identified. Interestingly, it was found that dotL mutants producing low levels of DotA were able to grow on agar plates. This important finding allowed considerable manipulation of phenotypes, because the behavior of mutants that would otherwise be inviable could now be evaluated during intracellular growth. The great majority of mutations, in fact, appeared to directly affect the assembly of the Dot/Icm system or disrupt function of DotL, and many of the impaired proteins are involved in either membrane biogenesis or membrane protein folding. In several T4SSs,Mob protein can be transferred into recipient cells in the absence of DNA mobilization, indicating that protein translocation into bacteria can occur in the same fashion that is observed when the pathogen contacts a mammalian cell. Furthermore, elimination of multiple paralogs of a single gene family does not solve the redundancy issue, suggesting that a similar amino acid sequence does not necessarily mean the proteins have similar functions. Development of functional and biochemical assays for these proteins will be necessary to sort out their roles in intracellular growth.
Citation:
R. Isberg R, Machner M.
2006.
Identification of Translocated Substrates of the Legionella pneumophila Dot/Icm System without the use of Eukaryotic Host Cells,
p 169-176.
In
Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed),
Legionella.
ASM Press, Washington, DC.
doi: 10.1128/9781555815660.ch45
Figures
Identification of Translocated Substrates of the Legionella pneumophila Dot/Icm System without the use of Eukaryotic Host Cells
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Citation:
R. Isberg R, Machner M.
2006.
Identification of Translocated Substrates of the Legionella pneumophila Dot/Icm System without the use of Eukaryotic Host Cells,
p 169-176.
In
Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed),
Legionella.
ASM Press, Washington, DC.
doi: 10.1128/9781555815660.ch45
/content/10.1128/9781555815660.ch45.ch45fig01
FIGURE 1
Isolation of lidA::miniTn10, a mutation that eliminates a translocated substrate of Dot/Icm (from [
7
]). (A) Insertion mutations were generated in an L. pneumophila strain that has a defective Dot/Icm transporter due to a mutation in the dotA gene. These strains were then patched onto bacteriological plates and allowed to grow. Strains of high viability were then patched onto a plate seeded with E. coli with a mobilizable plasmid that encodes an intact dotA
+ gene to allow mating and movement of the dotA gene into the mutagenized strains. Such strains will now have an intact Dot/Icm translo-cator, and should grow like wild-type strains unless the insertion mutation has caused a growth defect when there is a Dot/Icm system present. These mutants with lowered plating efficiency in the presence of an intact Dot/Icm will make a smaller patch on selective media than do strains that can tolerate an intact translocator. (B) Charcoal-yeast extract plates showing examples of mutants that fail to tolerate the presence of the Dot/Icm system.
10.1128/9781555815660/f0172-01_thmb.gif
10.1128/9781555815660/f0172-01.gif
FIGURE 1
Isolation of lidA::miniTn10, a mutation that eliminates a translocated substrate of Dot/Icm (from [ 7 ]). (A) Insertion mutations were generated in an L. pneumophila strain that has a defective Dot/Icm transporter due to a mutation in the dotA gene. These strains were then patched onto bacteriological plates and allowed to grow. Strains of high viability were then patched onto a plate seeded with E. coli with a mobilizable plasmid that encodes an intact dotA + gene to allow mating and movement of the dotA gene into the mutagenized strains. Such strains will now have an intact Dot/Icm translo-cator, and should grow like wild-type strains unless the insertion mutation has caused a growth defect when there is a Dot/Icm system present. These mutants with lowered plating efficiency in the presence of an intact Dot/Icm will make a smaller patch on selective media than do strains that can tolerate an intact translocator. (B) Charcoal-yeast extract plates showing examples of mutants that fail to tolerate the presence of the Dot/Icm system.
Citation:
R. Isberg R, Machner M.
2006.
Identification of Translocated Substrates of the Legionella pneumophila Dot/Icm System without the use of Eukaryotic Host Cells,
p 169-176.
In
Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed),
Legionella.
ASM Press, Washington, DC.
doi: 10.1128/9781555815660.ch45
/content/10.1128/9781555815660.ch45.ch45fig02
FIGURE 2
Identification of translocated substrates of Dot/Icm by transfer between bacterial strains (from [
13
]). Fusions of L. pneumophila DNA to the gene encoding Cre were constructed and were used to test the ability of the resulting hybrid protein to translocate into a tester L. pneumophila strain. Translocation of a hybrid protein from the donor strain can be detected by the generation of kanamycin-resistant (Kanr) exconjugants. These strains are recombinants that result from removal of a floxed transcriptional terminator located between a promoter and the kanr gene found on a construction in the recipient bacterial strain. In contrast, in the absence of Cre, bacteria harboring the intact reporter are unable to grow on media containing kanamycin and sucrose. The translocation of Cre hybrid protein into the recipient strain leads to the excision, via recombination at the loxP sites, of the DNA fragment that confers sucrose sensitivity (sacB) and reconstitution of a functional loxP-nptII translational fusion. S. D., Shine-Dalgarno sequence; nptII, neomycin phosphotransferase; ⋄, transcriptional terminator. Arrows indicate the trc promoter and loxP sites.
10.1128/9781555815660/f0173-01_thmb.gif
10.1128/9781555815660/f0173-01.gif
FIGURE 2
Identification of translocated substrates of Dot/Icm by transfer between bacterial strains (from [ 13 ]). Fusions of L. pneumophila DNA to the gene encoding Cre were constructed and were used to test the ability of the resulting hybrid protein to translocate into a tester L. pneumophila strain. Translocation of a hybrid protein from the donor strain can be detected by the generation of kanamycin-resistant (Kanr) exconjugants. These strains are recombinants that result from removal of a floxed transcriptional terminator located between a promoter and the kanr gene found on a construction in the recipient bacterial strain. In contrast, in the absence of Cre, bacteria harboring the intact reporter are unable to grow on media containing kanamycin and sucrose. The translocation of Cre hybrid protein into the recipient strain leads to the excision, via recombination at the loxP sites, of the DNA fragment that confers sucrose sensitivity (sacB) and reconstitution of a functional loxP-nptII translational fusion. S. D., Shine-Dalgarno sequence; nptII, neomycin phosphotransferase; ⋄, transcriptional terminator. Arrows indicate the trc promoter and loxP sites.
Citation:
R. Isberg R, Machner M.
2006.
Identification of Translocated Substrates of the Legionella pneumophila Dot/Icm System without the use of Eukaryotic Host Cells,
p 169-176.
In
Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed),
Legionella.
ASM Press, Washington, DC.
doi: 10.1128/9781555815660.ch45
References
/content/book/10.1128/9781555815660.ch45
1.
Beranek, A.,, M. Zettl,, K. Lorenzoni,, A. Schauer,, M. Manhart, and, G. Koraimann. 2004. Thirty-eight C-terminal amino acids of the coupling protein TraD of the F-like conjugative resistance plasmid R1 are required and sufficient to confer binding to the substrate selector protein TraM. J. Bacteriol. 186: 6999— 7006.
2.
Buscher, B. A.,, G. M. Conover,, J. L. Miller,, S. A. Vogel,, S. N. Meyers,, R. R. Isberg, and, J. P. Vogel. 2005. The DotL protein, a member of the TraG-coupling protein family, is essential for viability of Legionella pneumophila strain Lp02. J. Bacteriol. 187: 2927— 2938.
3.
Cabezon, E.,, J. I. Sastre, and, F. de la Cruz. 1997. Genetic evidence of a coupling role for the TraG protein family in bacterial conjugation. Mol. Gen. Genet. 254: 400— 406.
4.
Campodonico, E. M.,, L. Chesnel, and, C. R. Roy. 2005. A yeast genetic system for the identification and characterization of substrate proteins transferred into host cells by the Legionella pneumophila Dot/Icm system. Mol. Microbiol. 56: 918— 933.
5.
Chen, J.,, K. S. de Felipe,, M. Clarke,, H. Lu,, O. R. Anderson,, G. Segal, and, H. A. Shuman. 2004. Legionella effectors that promote nonlytic release from protozoa. Science 303: 1358— 1361.
6.
Christie, P. J., and, J. P. Vogel. 2000. Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends Mi-crobiol. 8: 354— 360.
7.
Conover, G. M.,, I. Derre,, J. P. Vogel, and, R. R. Isberg. 2003. The Legionella pneumophila LidA protein: a translocated substrate of the Dot/Icm system associated with maintenance of bacterial integrity. Mol. Microbiol. 48: 305— 321.
8.
Derre, I., and, R. R. Isberg. 2005. LidA, a translocated substrate of the Legionella pneumophila type IV secretion system, interferes with the early secretory pathway. Infect. Immun. 73: 4370— 4380.
9.
Kagan, J. C., and, C. R. Roy. 2002. Legionella phagosomes intercept vesicular traffic from endo-plasmic reticulum exit sites. Nat. Cell. Biol. 4: 945— 954.
10.
Karimova, G.,, J. Pidoux,, A. Ullmann, and, D. Ladant. 1998. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc. Natl. Acad. Sci. USA 95: 5752— 5756.
11.
Kirby, J. E.,, J. P. Vogel,, H. L. Andrews, and, R. R. Isberg. 1998. Evidence for pore-forming ability by Legionella pneumophila. Mol. Microbiol. 27: 323— 336.
12.
Lu, J., and, L. S. Frost. 2005. Mutations in the C-terminal region of TraM provide evidence for in vivo TraM-TraD interactions during F-plasmid conjugation. J. Bacteriol. 187: 4767— 4773.
13.
Luo, Z. Q., and, R. R. Isberg. 2004. Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer. Proc. Natl. Acad. Sci. USA 101: 841— 846.
14.
Marra, A.,, S. J. Blander,, M. A. Horwitz, and, H. A. Shuman. 1992. Identification of a Legionella pneumophila locus required for intracel-lular multiplication in human macrophages. Proc. Natl. Acad. Sci. USA 89: 9607— 9611.
15.
Nagai, H.,, J. C. Kagan,, X. Zhu,, R. A. Kahn, and, C. R. Roy. 2002. A bacterial guanine nu-cleotide exchange factor activates ARF on Legionella phagosomes. Science 295: 679— 682.
16.
Parsot, C.,, R. Menard,, P. Gounon, and, P. J. Sansonetti. 1995. Enhanced secretion through the Shigella flexneri Mxi-Spa translocon leads to assembly of extracellular proteins into macromo-lecular structures. Mol. Microbiol. 16: 291— 300.
17.
Russo, J. J., et al. 2001. Legionella Genome Project. Department of Microbiology, Columbia University. [Online.]
18.
Segal, G.,, M. Purcell, and, H. A. Shuman. 1998. Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome. Proc. Natl. Acad. Sci. USA 95: 1669— 1674.
19.
Shohdy, N.,, J. A. Efe,, S. D. Emr, and, H. A. Shuman. 2005. Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking. Proc. Natl. Acad. Sci. USA 102: 4866— 4871.
20.
Szpirer, C. Y.,, M. Faelen, and, M. Couturier. 2000. Interaction between the RP4 coupling protein TraG and the pBHR1 mobilization protein Mob. Mol. Microbiol. 37: 1283— 1292.
21.
Szpirer, C. Y.,, M. Faelen, and, M. Couturier. 2001. Mobilization function of the pBHR1 plas-mid, a derivative of the broad-host-range plasmid pBBR1. J. Bacteriol. 183: 2101— 2110.
22.
Tyndall, R. L., and, E. L. Domingue. 1982. Cocultivation of Legionella pneumophila and free-living amoebae. Appl. Environ. Microbiol. 44: 954— 959.
23.
Vergunst, A. C.,, B. Schrammeijer,, A. den Dulk-Ras,, C. M. de Vlaam,, T. J. Regensburg-Tuink, and, P. J. Hooykaas. 2000. VirB/D4-dependent protein translocation from Agrobac-terium into plant cells. Science 290: 979— 982.
24.
Vogel, J. P.,, H. L. Andrews,, S. K. Wong, and, R. R. Isberg. 1998. Conjugative transfer by the virulence system of Legionella pneumophila. Science 279: 873— 876.
25.
Winn, W. C., Jr., and, R. L. Myerowitz. 1981. The pathology of the Legionella pneumonias. A review of 74 cases and the literature. Hum. Pathol. 12: 401— 422.
Tables
Identification of Translocated Substrates of the Legionella pneumophila Dot/Icm System without the use of Eukaryotic Host Cells
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Citation:
R. Isberg R, Machner M.
2006.
Identification of Translocated Substrates of the Legionella pneumophila Dot/Icm System without the use of Eukaryotic Host Cells,
p 169-176.
In
Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed),
Legionella.
ASM Press, Washington, DC.
doi: 10.1128/9781555815660.ch45
/content/10.1128/9781555815660.ch45.ch45tab01
TABLE 1
Translocated substrates identified by the Cre-Lox interbacterial screen
TABLE 1
Translocated substrates identified by the Cre-Lox interbacterial screen
Citation:
R. Isberg R, Machner M.
2006.
Identification of Translocated Substrates of the Legionella pneumophila Dot/Icm System without the use of Eukaryotic Host Cells,
p 169-176.
In
Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed),
Legionella.
ASM Press, Washington, DC.
doi: 10.1128/9781555815660.ch45