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Infection and Immunity, January 2008, p. 170-178, Vol. 76, No. 1
0019-9567/08/$08.00+0     doi:10.1128/IAI.01913-06
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Mycobacterium avium subsp. paratuberculosis MAP3464 Gene Encodes an Oxidoreductase Involved in Invasion of Bovine Epithelial Cells through the Activation of Host Cell Cdc42{triangledown}

Marta Alonso-Hearn,1 Dilip Patel,2 Lia Danelishvili,1 Lisbeth Meunier-Goddik,3 and Luiz E. Bermudez1*

Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, Oregon 97331,1 Food Safety Center of Excellence, University of Tennessee, Knoxville, Tennessee 37996,2 Department of Food Science, Oregon State University, Corvallis, Oregon 973313

Received 4 December 2006/ Returned for modification 19 February 2007/ Accepted 28 September 2007

Mycobacterium avium subsp. paratuberculosis infection of cattle takes place through the intestinal mucosa. To identify M. avium subsp. paratuberculosis genes associated with the invasion of bovine epithelial cells in vitro, we screened a library of transposon mutants. Several mutants of M. avium subsp. paratuberculosis were identified which invaded Madin-Darby bovine kidney (MDBK) epithelial cells less efficiently than wild-type (wt) M. avium subsp. paratuberculosis. The {Delta}Ox mutant had the transposon located in the MAP3464 gene, a putative oxidoreductase gene whose expression is upregulated upon bacterial contact with MDBK cells. Complete restoration of invasion comparable to that for the wt bacterium was achieved by introducing a copy of the complete oxidoreductase operon into the {Delta}Ox mutant. Immunoprecipitation and Western blot analysis indicated that wt M. avium subsp. paratuberculosis activates Cdc42 and RhoA pathways of internalization 15 and 60 min after infection of the host cell, respectively. The {Delta}Ox mutant, however, failed to activate the Cdc42 pathway. To determine whether an M. avium subsp. paratuberculosis protein delivered to the host cell mediates the entry of the wt bacterium by activation of the Cdc42 pathway, affinity precipitation of active Cdc42 from MDBK-infected cells followed by mass spectrometry was carried out. We identified a 17-amino-acid bacterial peptide associated with the Cdc42 of cells infected with wt M. avium subsp. paratuberculosis but not with the {Delta}Ox mutant. The sequence of the peptide matches MAP3985c, a hypothetical protein, possibly functioning as a putative Cdc42 effector. These findings reveal a novel signaling pathway activated during M. avium subsp. paratuberculosis entry that links the product of MAP3464 gene to activation of Cdc42 in the host cell.

* Corresponding author. Mailing address: Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, 100 Magruder Hall, Corvallis, OR 97331-4802. Phone: (541) 737-6538. Fax: (541) 737-2730. E-mail: Luiz.Bermudez{at}oregonstate.edu

{triangledown} Published ahead of print on 15 October 2007.

Editor: J. F. Urban, Jr.

Infection and Immunity, January 2008, p. 170-178, Vol. 76, No. 1
0019-9567/08/$08.00+0     doi:10.1128/IAI.01913-06
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Harriff, M. J., Danelishvili, L., Wu, M., Wilder, C., McNamara, M., Kent, M. L., Bermudez, L. E. (2009). Mycobacterium avium Genes MAV_5138 and MAV_3679 Are Transcriptional Regulators That Play a Role in Invasion of Epithelial Cells, in Part by Their Regulation of CipA, a Putative Surface Protein Interacting with Host Cell Signaling Pathways. J. Bacteriol. 191: 1132-1142 [Abstract] [Full Text]  


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