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P2-189 Use of a Proteomic and Genotypic Approach to Understand Proteins Associated with Outer Membrane Vesicles Secreted by Cronobacter Species

Monday, July 27, 2015
Exhibit Hall (Oregon Convention Center)
Mahendra Kothary , U.S. Food and Drug Administration , Laurel , MD
Prasad Rallabhandi , U.S. Food and Drug Administration , Laurel , MD
Michael Kulka , U.S. Food and Drug Administration , Laurel , MD
Qiongqiong Yan , University College Dublin , Dublin , Ireland
Hannah Chase , U.S. Food and Drug Administration , Laurel , MD
Eunbi Park , U.S. Food and Drug Administration , Laurel , MD
TaeJung Chung , U.S. Food and Drug Administration , Laurel , MD
YeonJoo Yoo , U.S. Food and Drug Administration , Laurel , MD
Laurenda Carter , U.S. Food and Drug Administration , Laurel , MD
Gopal Gopinath , U.S. Food and Drug Administration , Laurel , MD
Seamus Fanning , University College Dublin , Dublin , Ireland
Ben D. Tall , U.S. Food and Drug Administration , Laurel , MD
Introduction: Secretion of outer membrane (OM) vesicles (OMVs) by Gram-negative bacteria occurs under in vitro and in vivo growth conditions.  OMVs are thought to contribute to pathogenesis by delivering LPS components, OM proteins (OMPs), and effector molecules such as toxins, enzymes, adhesins, and nucleic acids to target host cells. Known examples include the ompA protein and the heat-labile enterotoxin expressed by Acinetobacter baumannii and Escherichia coli, respectively.

Purpose: However, the secretion of OMVs by Cronobacter and their contribution to pathogenesis is unknown. In this study, OMVs were obtained from C. sakazakii, C. malonaticus, and C. turicensis and their proteome-associated contents were analyzed by SDS-PAGE, and protein sequencing.

Methods: OMVs were partially purified from cells grown on Trypticase soy agar with 1% NaCl at 37°C for 18 h by removing the cells by centrifugation (8,000 vg, 15 min.). OMVs in the supernatant were concentrated by ultracentrifugation, and the resulting pellets were analyzed by SDS-PAGE. Separated proteins were transferred to membranes, and stained with Coomassie blue.  Protein bands were excised and sequenced.  PCR primers targeting OMV genes were designed and used for determining their presence in a collection of 240 strains.

Results: Electron microscopy of negatively-stained cells showed that the OMVs are secreted as pleomorphic microvesicles (< 300 nm in size).  Sequence analysis indicated that OMVs contained proteins with strong homologies to OmpA, OmpC, OmpX, MipA, conjugative plasmid transfer protein (CTP), GroEL, and an OM autotransporter/adhesin (OMATP) protein. PCR analysis revealed that these genes are common among Cronobacter species. However, in comparison to strains representing other phylogenetically-related species, gene targets such as the CTP gene and OMATP were found to be Cronobacter-specific.

Significance: The presence of OMPs and their genes suggests that OMVs in Cronobacter are involved in multiple functions including adherence and invasion, stress response, plasmid maintenance, and extracellular transport, alluding to their possible roles in pathogenesis.

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