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Chapter 2 : Role of the Type II Protein Secretion Pathway in Pathogenesis of

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Role of the Type II Protein Secretion Pathway in Pathogenesis of , Page 1 of 2

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Abstract:

To test whether the phenotype of the mutant is due to loss of the type II protein secretion system and/or the type IV pilus assembly apparatus, the authors employed a genetic approach. First, the authors mutated two loci involved in type II protein secretion. Second, the authors analyzed the phenotypes of two mutants defective in the biogenesis of type IV pilus. The protease, acid phosphatase, and pnitrophenyl phosphorylcholine (PNPPC) hydrolase activities were similarly reduced in the supernatant of the , , and mutants compared with the wild type and the and mutant strains. In addition, the lipase and phospholipase A activities were also reduced in the supernatants of both mutants relative to the wild type and the mutant. These results demonstrate that five -dependent activities are secreted by the type II protein secretion pathway. Indeed, the small growth defect exhibited by some mutants in U937 cells could hint at a minor role of the type IV pilus assembly apparatus itself for macrophage infection. This defect, added to the small defect due to the loss of type II secretion, could account for the growth impairment of the mutant in macrophages. Alternatively, could control a third unidentified pathway promoting macrophage infection. Thus, continued analysis of , , and pilus mutants should not only expand our understanding of Legionnaires’ disease but may provide new paradigms for protein secretion systems.

Citation: Rossier O, Cianciotto N, Edelstein P. 2002. Role of the Type II Protein Secretion Pathway in Pathogenesis of , p 13-17. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch2

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Figures

Image of FIGURE 1
FIGURE 1

Secreted enzymatic activities of strains. Late logarithmic phase supernatants of wild-type 130b, , , , , and mutants were tested for (A) protease activity as determined by azocasein hydrolysis; (B) acid phosphatase activity as determined by the release of -nitrophenyl (PNP) from PNP phosphate at pH 5.0; (C) PNPPC hydrolysis; (D) lipase activity as determined by the release of free fatty acid (FFA) from 1-monopalmitoyl glycerol; and (E) phospholipase A activity as determined by phosphatidylcholine hydrolysis. These data represent the mean and standard deviation for duplicate cultures. For all, the difference between the wild type and the and mutants were significant (P < 0.01, Student's t test). Note that the mutant was not tested for the lipolytic activities. Comparable results were obtained on at least two other occasions (data not shown).

Citation: Rossier O, Cianciotto N, Edelstein P. 2002. Role of the Type II Protein Secretion Pathway in Pathogenesis of , p 13-17. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch2
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Image of FIGURE 2
FIGURE 2

Intracellular infection of amoebae with strains. Wells containing were infected at a multiplicity of infection of 0.1 with wild-type 130b (black diamonds), mutant (black squares), mutant (shaded circles), mutant (shaded triangles), and mutant (white squares). Bacterial CFU per well were determined at 0, 24, 48, and 72 h after inoculation. Each datum point represents the mean and standard deviation of three wells. With the exception of the mutant, significant differences in recovery between 130b and its mutant derivatives were evident at 48 h ( < 0.01, Student's test). These differences were observed in three additional experiments (data not shown).

Citation: Rossier O, Cianciotto N, Edelstein P. 2002. Role of the Type II Protein Secretion Pathway in Pathogenesis of , p 13-17. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch2
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Image of FIGURE 3
FIGURE 3

Macrophage infection by strains. (A) U937 cells were infected at a multiplicity of infection of 0.1 with 130b (black diamonds), mutant (black squares), mutant (gray triangles), and mutant (white squares). (B) U937 cells were infected at a multiplicity of infection of 0.1 with 130b (black diamonds), mutant 1 (white triangles), and mutant 2 (gray squares). Two mutants were comparable to 1, while another was like 2. Bacterial CFU per monolayers were determined at 0, 24, and 48 h after inoculation. Each datum point represents the mean and standard deviation of three wells. Unlike the mutant, significant differences in recovery between 130b and the mutant were evident at 48 h ( < 0.001, Student's test). Similar results were obtained for the mutant and were observed in three additional experiments (data not shown).

Citation: Rossier O, Cianciotto N, Edelstein P. 2002. Role of the Type II Protein Secretion Pathway in Pathogenesis of , p 13-17. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch2
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Image of FIGURE 4
FIGURE 4

Model for the contributions of type II protein secretion, type IV pilus, and prepilin peptidase PilD to intracellular infection. IM, inner membrane; OM, outer membrane. For more details see text.

Citation: Rossier O, Cianciotto N, Edelstein P. 2002. Role of the Type II Protein Secretion Pathway in Pathogenesis of , p 13-17. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch2
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References

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1. Aragon, V.,, S. Kurtz,, A. Flieger,, B. Neu-meister,, and N. P. Cianciotto. 2000. Secreted enzymatic activities of wild-type and pilD-deficient Legionella pneumophila. Infect. Immun. 68:18551863.
2. Hales, L. M.,, and H. A. Shuman. 1999. Legionella pneumophila contains a type II general secretion pathway required for growth in amoebae as well as for secretion of the Msp protease. Infect. Immun. 67:36623666.
3. Liles, M. R.,, P. H. Edelstein,, and N. P. Cianciotto. 1999. The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila. Mol. Microbiol. 31:959970.
4. Russel, M. 1998. Macromolecular assembly and secretion across the bacterial cell envelope: type II protein secretion systems. J. Mol. Biol. 279:485499.
5. Stone, B. J.,, and Y. Abu Kwaik. 1998. Expression of multiple pili by Legionella pneumophila: identification and characterization of a type IV pi-lin gene and its role in adherence to mammalian and protozoan cells. Infect. Immun. 66:17681775.

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