Chapter 15 : Mechanisms of Entry into Mammalian Cells

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To begin to examine the process whereby the enteric pathogenic yersiniae enter the host, in 1981 Devenish and Schiemann developed the now famous invasion assay, an in vitro model for the entry of into mammalian cells. The introduced sequences are expressed, and Ail protein can be detected on the bacterial cell surface. One explanation for the absence of Ail-mediated entry in these nonpathogenic strains containing the gene is that other factors, in addition to Ail, that affect yersinia entry are required. Two different virulence factors of have been identified as being homologous to Ail. All three genes , , described have been identified as being able to promote bacterial entry into mammalian cells in vitro. isolates do not enter mammalian cells in vitro, however. However, it is known that when the gene from is introduced into , the resulting strain is able to enter mammalian cells. Both Ail and YadA have been shown to mediate other phenotypes in spp., including resistance to serum killing. This apparent redundancy in function may yet turn out to be an in vitro artifact or may instead have evolved to allow the bacterium to survive in the myriad of environments it encounters during the normal course of infection.

Citation: Pierson D. 1994. Mechanisms of Entry into Mammalian Cells, p 235-247. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch15

Key Concept Ranking

Outer Membrane Proteins
Peyer's Patches
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Table 1

Enrichment procedure yields strains that invade cell culture monolayers

From Isberg, R. R., and S. Falkow. 1985. (London) 317:262-264 ( ). Reprinted with permission from (London) 317:262−264. Copyright 1985 Macmillan Magazines Limited. Saturated cultures of bacteria, grown at 28°C, were washed twice in phosphate-buffered saline (PBS) and resuspended to a concentration of 3 × 10 bacteria ml. Aliquots of 50 μl of each strain were added to monolayer cultures of HEp-2 cells seeded at a concentration of 2 × 10 animal cells per microtiter well (24-well Falcon 3047 microliter dishes) in RPMI 1640 medium. Bacteria were centrifuged onto the monolayer at 600 × as described previously ( ), and the infected cultures were incubated at 36°C for 3 h in a 5% CO atmosphere, to allow binding and invasion of bacteria. Nonadherent bacteria were removed from the monolayer by washing three times with sterile PBS, and RPMI 1640 medium containing 40 μg of gentamicin (Sigma) ml was added to each microtiter well. The incubation was continued at 36°C for 2 h in the presence of the antibiotic before washing of the monolayers in PBS twice more. Internalized bacteria were then released from the monolayers by the addition of 1% Triton X-100, and titers were determined on L-agar plates ( ).

Percentage of bacteria added to HEp-2 monolayers that resist treatment by gentamicin.

strain ( ).

K-12 strain HB101.

HB101 harboring cosmids that are denoted in parentheses.

Citation: Pierson D. 1994. Mechanisms of Entry into Mammalian Cells, p 235-247. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch15
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Table 2

Relative entry of clones into HEp-2 cells

Strains were used to infect a monolayer of the human larynx epithelial cell line HEp-2. Percent invasion is as described in footnote of Table 1 . Adapted from Miller, V. L., and S. Falkow. 1988. 56:1242-1248 ( ).

Citation: Pierson D. 1994. Mechanisms of Entry into Mammalian Cells, p 235-247. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch15
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Table 3

YadA-mediated entry of into HEp-2 cells

Percent invasion was determined as described in footnote of Table 1 . Adapted from Yang, Y., and R. Isberg. 1993. 61:3907−3913 ( ).

Citation: Pierson D. 1994. Mechanisms of Entry into Mammalian Cells, p 235-247. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch15

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