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Chapter 26 : Pore Formation-Mediated Egress from Mammalian and Protozoan Cells by

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Pore Formation-Mediated Egress from Mammalian and Protozoan Cells by , Page 1 of 2

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Abstract:

Infections of mammalian and protozoan cells by share a number of similarities but differ in several attributes. The mechanisms of killing of the host cell and release of intracellular bacteria after termination of intracellular replication are not known for or any other vacuolar intracellular pathogen. During screening of the mini Tn::kan mutant library the intracellular bacteria, , belonging to the parental strain AA100 were released into the tissue culture medium with in 24 to 48 h postinfection. In contrast, despite the prolific intracellular replication of the five mutants, they were "trapped" within and failed to egress from macrophages and epithelial cells during the 48-h infection, and the majority of the infected cells remained viable and intact. Researchers have recently proposed a model of biphasic death of mammalian cells by initiated by caspase-3-dependent apoptosis followed by necrosis, which is probably mediated by the pore-forming toxin. To confirm the roles played by the pore-forming activity in necrotic killing of and subsequent release of the intracellular bacteria, researchers examined infections at a multiplicity of infection (MOI) of 500, for the following two reasons. First, infection at this MOI may ensure that most of the cells were infected by and may allow better examination of whether the rib mutants were defective in exiting the protozoan host. Second, it has been shown that extracellular induces rapid necrotic killing of mammalian cells within 20 to 180 min at an MOI of 500.

Citation: Terry Alii O, Molmoret M, Abu Kwaik Y. 2002. Pore Formation-Mediated Egress from Mammalian and Protozoan Cells by , p 143-151. 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.ch26

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Figures

Image of FIGURE 1
FIGURE 1

The mutants are defective in killing and exiting U937 macrophages, but not in intracellular replication. (A) Cytopathogenicity to infected cells (MOI 5) was determined by Alamar blue assays and compared with that of the noninfected cells. (B) Growth kinetics within U937 macrophages, where the indicated number of bacteria is the combined numbers of intracellular bacteria and the ones that were released into the supernatant. (C) The bacteria that were released into the tissue culture medium. (D) The number of intracellular bacteria. Values are the mean of triplicate samples, and error bars represent standard deviations. Reprinted from reference with permission.

Citation: Terry Alii O, Molmoret M, Abu Kwaik Y. 2002. Pore Formation-Mediated Egress from Mammalian and Protozoan Cells by , p 143-151. 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.ch26
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Image of FIGURE 2
FIGURE 2

The mutants defect in cytolysis of the host cell is due to a defect in necrosis-mediated killing. Representative transmission electron micrographs of infected U937 macrophages at 24 h and 48 h postinfection by the wild-type strain AA100 and the GN229 mutant. Magnifications are ×7.000 and ×5,000 for the 24-h and 48-h infections, respectively. Reprinted from reference with permission.

Citation: Terry Alii O, Molmoret M, Abu Kwaik Y. 2002. Pore Formation-Mediated Egress from Mammalian and Protozoan Cells by , p 143-151. 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.ch26
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Image of FIGURE 3
FIGURE 3

Representative phase contrast images of infected by the wild-type strain AA100 or the GN229 mutant of and compared with noninfected (N1) cells. Infections were performed at an MOI of 1, exactly as described in the Fig. 5 legend. Phase contrast images at 4, 24, and 48 h postinfection are shown. Similar results to the GN229 mutant were also obtained for the GP247 mutant (data not shown). Reprinted from reference with copyright permission from Blackwell Sciences.

Citation: Terry Alii O, Molmoret M, Abu Kwaik Y. 2002. Pore Formation-Mediated Egress from Mammalian and Protozoan Cells by , p 143-151. 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.ch26
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Image of FIGURE 4
FIGURE 4

Growth phase-dependent expression of the pore-forming activity by , in vitro and intracellularly. Contact-dependent hemolysis of SRBCs by in vitro-grown (A) or intracellular bacteria, isolated from U937 macrophages (?). Infection of the cells in panel ? was performed using in vitro-grown bacteria that reached their maximal growth and hemolysis (14 h in panel A). At the indicated time points, the bacterial growth was determined by the absorbance at 550 nm (A) in panel A or by the CFU in panel ? (left axis), and hemolytic activity was determined (right axis) using equivalent numbers of bacteria at all time points. Values are the mean of triplicate samples, and error bars represent standard deviations. Bact., bacterial. Reprinted from reference with permission.

Citation: Terry Alii O, Molmoret M, Abu Kwaik Y. 2002. Pore Formation-Mediated Egress from Mammalian and Protozoan Cells by , p 143-151. 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.ch26
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Image of FIGURE 5
FIGURE 5

A model of growth phase-dependent cytolysis of mammalian cells by upon termination of intracellular bacterial replication to egress from the spent host cell. During early stages of formation of the mitochondria and RER-surrounded phagosome (A) and during exponential intracellular replication (B) expression of the Rib toxin is turned off, but caspase-3-mediated apoptosis is triggered. Upon transition to the postexponential phase of growth, the Rib toxin activity is triggered, which results in insertions of pores in the phagosomal membrane first (C), leading to its disruption (D); see Fig. 1 for the absence of a recognizable phagosomal membrane. This is followed by insertions of the pores in the plasma membrane (E), leading to osmotic lysis of the ??? and release of the intracellular bacteria. Reprinted from reference with permission. The same model applies to protozoa with the exception of the absence of apoptosis.

Citation: Terry Alii O, Molmoret M, Abu Kwaik Y. 2002. Pore Formation-Mediated Egress from Mammalian and Protozoan Cells by , p 143-151. 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.ch26
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References

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