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Chapter 63 : The Hsp60 Chaperonin of : an Intriguing Player in Infection of Host Cells

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Abstract:

This chapter reports that through genetic and functional studies in yeast and mammalian cells, the authors have demonstrates that Hsp60 alters signaling cascades in eukaryotic cells, modifies the actin cytoskeleton of mammalian cells, and alters the trafficking of mitochondria in mammalian cells, making Hsp60 a potential effector in ’s intracellular establishment. The authors determined that and mutant derivatives of Lp02 had virtually no protease-accessible HtpB; i.e., these mutants displayed no surface-exposed HtpB. In addition, immunogold electron microscopy, performed with an HtpB-specific rabbit antiserum and a secondary gold-conjugated antibody against rabbit immunoglobulin, showed that the Lp02 dot mutants, particularly the mutant, had an increased amount of periplasmic gold particles. This interesting result suggested that a nonfunctional Dot/Icm system results in the accumulation of HtpB in the periplasm. Recombinant HtpB caused Chinese hamster ovary (CHO) cells to lose their stress fibers. The abilities of HtpB to specifically alter eukaryotic signaling pathways, cytoskeletal organization, and organellar traffic are indeed functional characteristics that fit well into HtpB’s potential role as an virulence effector.

Citation: Chong A, Riveroll A, S. Allan D, Garduño E, A. Garduño R. 2006. The Hsp60 Chaperonin of : an Intriguing Player in Infection of Host Cells, p 255-260. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch63

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Bacterial Proteins
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Type IV Secretion Systems
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Figures

Image of FIGURE 1
FIGURE 1

Immunoblots of sodium dodecylsulfate-polyacrylamide gel eclectrophoresis-resolved proteins from whole bacterial cells treated with trypsin (250 μg trypsin/10 bacteria) and immunostained with an HtpB-specific monoclonal antibody and alkaline phosphatase-conjugated rabbit antimouse IgG. () Band patterns produced in the wild-type strain Lp02 and its two derivatives JV303 mutant) and JV309 mutant). The bracket labeled with a D marks the region where HtpB degradation products appear, and the hollow arrowheads point at full-size nondegraded HtpB. Band patterns showing a clear degradation of trypsin-accessible HtpB in the virulent strain 2064 (used as a positive control for the assay [ ]) and the lack of trypsin-accessible HtpB in the 2064M salttolerant mutant (used as a negative control for the assay [ ]). C, control, trypsin-free sample; T, trypsintreated sample. The hollow arrowhead points at full-size nondegraded HtpB. Black arrows at the left side of both panels point to the position of three of the broad-range prestained protein size markers (New England Biolabs): from top to bottom, 62-, 47-, and 37.5-kDa, respectively.

Citation: Chong A, Riveroll A, S. Allan D, Garduño E, A. Garduño R. 2006. The Hsp60 Chaperonin of : an Intriguing Player in Infection of Host Cells, p 255-260. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch63
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Image of FIGURE 2
FIGURE 2

Immunoelectron microscopy of ultra-thin sections of Lp02 derivative JV303 mutant). Sectioned bacteria were labeled with HtpB-specific polyclonal antibody and antirabbit goat IgG conjugated with gold spheres. Gold spheres are ∼10 nm in diameter and appear as well-defined black dots on the micrograph. Notice the abundant clustering-banding of gold particles at the periphery of the bacterial sections, corresponding to the bacterial cell envelope and periplasmic regions. Labeling of the Lp02 parent strain results in a single row of gold particles lining the outer membrane (not shown), but not in clustering of abundant gold particles. The size bar represents 100 nm.

Citation: Chong A, Riveroll A, S. Allan D, Garduño E, A. Garduño R. 2006. The Hsp60 Chaperonin of : an Intriguing Player in Infection of Host Cells, p 255-260. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch63
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Image of FIGURE 3
FIGURE 3

Effects of HtpB on mammalian cells as evaluated through fluorescence microscopy ( and ) or transmission electron microscopy (). Panels a and b are images of CHO cells stained with fluorescently labeled phalloidin (which binds to polymerized actin), taken in a Zeiss confocal microscope. CHO cells normally show numerous stress fibers (clearly seen as bright lines going across the CHO cell in panel a. When expressed as a recombinant protein in the cytoplasm of CHO cells, HtpB causes the disappearance of actin stress fibers as shown in panel b. In addition, HtpB causes the appearance of peripheral bundles of polymerized actin (e.g., white arrowhead in panel b), an effect that we have called “framing.” (c) Electron micrograph of a sectioned CHO cell showing an internalized HtpB-coated microsphere (black arrow) surrounded by numerous mitochondria. Notice that some mitochondria (e.g., white arrowhead) come in close apposition to the membrane surrounding the internalized microsphere.

Citation: Chong A, Riveroll A, S. Allan D, Garduño E, A. Garduño R. 2006. The Hsp60 Chaperonin of : an Intriguing Player in Infection of Host Cells, p 255-260. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch63
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