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Open Access Highly Accessed Research article

Phospholipase D promotes Arcanobacterium haemolyticum adhesion via lipid raft remodeling and host cell death following bacterial invasion

Erynn A Lucas14, Stephen J Billington15, Petteri Carlson2, David J McGee3* and B Helen Jost16

Author Affiliations

1 Department of Veterinary Science and Microbiology, The University of Arizona, 1117 E Lowell Street, Tucson, AZ 85721, USA

2 Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Haartmaninkatu 3, P.O. Box 21, Helsinki, FIN-00014, Finland

3 Department of Microbiology and Immunology, Louisiana State University Health Sciences Center-Shreveport, 1501 Kings Highway, Shreveport, LA 71130, USA

4 Modesto Junior College, 435 College Avenue, Modesto, CA 95350, USA

5 Ventana Medical Systems, Inc., 1910 Innovation Park Drive, Oro Valley, AZ 85755, USA

6 Office for the Responsible Conduct of Research, The University of Arizona, P.O. Box 245092, Tucson, AZ 85721, USA

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BMC Microbiology 2010, 10:270  doi:10.1186/1471-2180-10-270

Published: 25 October 2010

Abstract

Background

Arcanobacterium haemolyticum is an emerging bacterial pathogen, causing pharyngitis and more invasive infections. This organism expresses an unusual phospholipase D (PLD), which we propose promotes bacterial pathogenesis through its action on host cell membranes. The pld gene is found on a genomic region of reduced %G + C, suggesting recent horizontal acquisition.

Results

Recombinant PLD rearranged HeLa cell lipid rafts in a dose-dependent manner and this was inhibited by cholesterol sequestration. PLD also promoted host cell adhesion, as a pld mutant had a 60.3% reduction in its ability to adhere to HeLa cells as compared to the wild type. Conversely, the pld mutant appeared to invade HeLa cells approximately two-fold more efficiently as the wild type. This finding was attributable to a significant loss of host cell viability following secretion of PLD from intracellular bacteria. As determined by viability assay, only 15.6% and 82.3% of HeLa cells remained viable following invasion by the wild type or pld mutant, respectively, as compared to untreated HeLa cells. Transmission electron microscopy of HeLa cells inoculated with A. haemolyticum strains revealed that the pld mutant was contained within intracellular vacuoles, as compared to the wild type, which escaped the vacuole. Wild type-infected HeLa cells also displayed the hallmarks of necrosis. Similarly inoculated HeLa cells displayed no signs of apoptosis, as measured by induction of caspase 3/7, 8 or 9 activities.

Conclusions

These data indicate that PLD enhances bacterial adhesion and promotes host cell necrosis following invasion, and therefore, may be important in the disease pathogenesis of A. haemolyticum infections.