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Infection and Immunity, November 1999, p. 5775-5783, Vol. 67, No. 11
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Outer Membrane Protein A-Promoted Actin Condensation of Brain Microvascular Endothelial Cells Is Required for Escherichia coli Invasion

Nemani V. Prasadarao,1,2,* Carol A. Wass,1 Monique F. Stins,1 Hiroyuki Shimada,2,3 and Kwang Sik Kim1,2

Division of Infectious Diseases1 and Department of Pathology,3 Childrens Hospital Los Angeles, and University of Southern California School of Medicine,2 Los Angeles, California 90027

Received 20 May 1999/Returned for modification 29 July 1999/Accepted 26 August 1999

Escherichia coli is the most common gram-negative bacterium that causes meningitis during the neonatal period. We have previously shown that the entry of circulating E. coli organisms into the central nervous system is due to their ability to invade the blood-brain barrier, which is composed of a layer of brain microvascular endothelial cells (BMEC). In this report, we show by transmission electron microscopy that E. coli transmigrates through BMEC in an enclosed vacuole without intracellular multiplication. The microfilament-disrupting agents cytochalasin D and latrunculin A completely blocked E. coli invasion of BMEC. Cells treated with the microtubule inhibitors nocodazole, colchicine, vincristin, and vinblastine and the microtubule-stabilizing agent taxol also exhibited 50 to 60% inhibition of E. coli invasion. Confocal laser scanning fluorescence microscopy showed F-actin condensation associated with the invasive E. coli but no alterations in microtubule distribution. These results suggest that E. coli uses a microfilament-dependent phagocytosis-like endocytic mechanism for invasion of BMEC. Previously we showed that OmpA expression significantly enhances the E. coli invasion of BMEC. We therefore examined whether OmpA expression is related to the recruitment of F-actin. OmpA+ E. coli induced the accumulation of actin in BMEC to a level similar to that induced by the parental strain, whereas OmpA- E. coli did not. Despite the presence of OmpA, a noninvasive E. coli isolate, however, did not show F-actin condensation. OmpA+-E. coli-associated condensation of F-actin was blocked by synthetic peptides corresponding to the N-terminal extracellular domains of OmpA as well as BMEC receptor analogues for OmpA, chitooligomers (GlcNAcbeta 1-4GlcNAc oligomers). These findings suggest that OmpA interaction is critical for the expression or modulation of other bacterial proteins that will subsequently cause actin accumulation for the uptake of bacteria.

* Corresponding author. Mailing address: Division of Infectious Diseases, Mail Stop no. 51, Childrens Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027. Phone: (323) 669-5622. Fax: (323) 660-2661. E-mail: pnemani{at}chla.usc.edu.

Infection and Immunity, November 1999, p. 5775-5783, Vol. 67, No. 11
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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