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Infect Immun, June 1998, p. 2928-2937, Vol. 66, No. 6
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Physical Limitations on Salmonella typhi Entry into Cultured Human Intestinal Epithelial Cells

Xiao-Zhe Huang,1 Ben Tall,2 William R. Schwan,1,dagger and Dennis J. Kopecko1,*

Laboratory of Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892,1 and Division of Microbiological Studies, CFSAN, Food and Drug Administration, Washington, DC 202042

Received 27 August 1997/Returned for modification 6 November 1997/Accepted 5 March 1998

Kinetic studies of Salmonella typhi invasion of INT407 cells at different multiplicities of infection (MOIs) have revealed a strict physical limitation on S. typhi entry at MOIs of >= 40. Staining of infected monolayers to distinguish intracellular from extracellular bacteria revealed that all monolayer cells are susceptible to infection and that internalized bacteria are typically contained in one to three separate clusters per cell during the first 60 min. Scanning and transmission electron microscopic analyses of time course-infected monolayers showed that at early times postinfection, bacteria bind to shortened, coalesced microvilli in one to three focal aggregate structures per host cell surface. As reported previously for S. typhimurium, focal aggregates progress to conical membrane ruffles that appear to engulf one or a few centrally contained S. typhi cells by a macropinocytic process, which enhanced the entry of simultaneously added Escherichia coli HB101 about 30-fold. Additionally, kinetic studies showed that at an MOI of sime 400, maximal S. typhi entry is virtually completed within 30 to 35 min. Monolayers pretreated with S. typhi for 30 min to saturate the entry process were severely reduced in the ability to internalize subsequently added kanamycin-resistant strains of S. typhi or S. typhimurium, but E. coli HB101(pRI203) expressing the cloned Yersinia inv gene was not reduced in entry. In invasion inhibition assays, anti-beta 1 integrin antibodies markedly reduced E. coli HB101(pRI203) invasion efficiency but did not reduce S. typhi entry. Collectively, these data provide direct physical and visual evidence which indicates that S. typhi organisms are internalized at a limited number (i.e., two to four) of sites on host cells. S. typhi and S. typhimurium likely share INT407 cell entry receptors which do not appear to be members of the beta 1 integrin superfamily.

* Corresponding author. Mailing address: Laboratory of Enteric and STD's, HFM 440, FDA-CBER, Bldg. 29, NIH Campus, Bethesda, MD 20892. Phone: (301) 496-1893. Fax: (301) 402-2776. E-mail: KOPECKO{at}A1.CBER.FDA.GOV.

dagger Present address: Pathogenesis Corp., West Seattle, WA 98119.

Infect Immun, June 1998, p. 2928-2937, Vol. 66, No. 6
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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