Description of a novel adhesin of Mycobacterium avium subsp. paratuberculosis.

Mariana Noelia Viale,María Isabel Romano,Gabriela Echeverria-Valencia,Marisa Fernandez,Pablo Romasanta,Emilio Malchiodi,María Laura Mon,María De La Paz Santangelo,Andrea Karina Gioffré

doi:10.1155/2014/729618

Abstract

The binding and ingestion of Mycobacterium avium subsp. paratuberculosis (MAP) by host cells are fibronectin (FN) dependent. In several species of mycobacteria, a specific family of proteins allows the attachment and internalization of these bacteria by epithelial cells through interaction with FN. Thus, the identification of adhesion molecules is essential to understand the pathogenesis of MAP. The aim of this study was to identify and characterize FN binding cell wall proteins of MAP. We searched for conserved adhesins within a large panel of surface immunogenic proteins of MAP and investigated a possible interaction with FN. For this purpose, a cell wall protein fraction was obtained and resolved by 2D electrophoresis. The immunoreactive spots were identified by MALDI-TOF MS and a homology search was performed. We selected elongation factor Tu (EF-Tu) as candidate for further studies. We demonstrated the FN-binding capability of EF-Tu using a ligand blot assay and also confirmed the interaction with FN in a dose-dependent manner by ELISA. The dissociation constant of EF-Tu was determined by surface plasmon resonance and displayed values within the μM range. These data support the hypothesis that this protein could be involved in the interaction of MAP with epithelial cells through FN binding.

Highlights

Paratuberculosis (PTB) is a chronic granulomatous enteritis of domestic and wild ruminants
Fibronectin (FN) binding is required for attachment and internalization of Mycobacterium avium subsp. paratuberculosis (MAP) by the epithelial cells and β1 integrins have been identified as the host cell receptors for FN-opsonized mycobacteria in vitro and in vivo [4]
We selected elongation factor Tu (EF-Tu) because the homologous of Mycoplasma pneumoniae and Acinetobacter baumannii functions as a FN-binding protein that facilitates the interactions between bacteria and extracellular matrix [30,31,32]

Summary

Introduction

Paratuberculosis (PTB) is a chronic granulomatous enteritis of domestic and wild ruminants. The members of this complex are found within the outer envelope and culture supernatants of mycobacteria and are immunodominant antigens [16, 17] These proteins possess mycolyltransferase activity and catalyze the synthesis of the most abundant glycolipid of the mycobacterial cell wall, trehalose 6,6-dimycolate (TDM) [18]. FAP is a member of a family of FNbinding proteins present in several species of mycobacteria that mediate the attachment and internalization of these bacteria by epithelial cells in vitro [19,20,21,22,23] This protein is called APA (for alanine-proline-rich antigen) and is encoded by a gene annotated as MAP1569 in the MAP K-10 strain. Other cell wall proteins could interact with FN to facilitate complex formation and, in this way, allow adherence to epithelial cells

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