The recovery of Mycobacterium avium subspecies paratuberculosis from the intestine of infected ruminants for proteomic evaluation Sharon Egan a,b, 1 , Mark Lanigan a, 1 , Brian Shiell a , Gary Beddome a , David Stewart a , Jill Vaughan a , Wojtek P. Michalski a, ⁎ a Protein Biochemistry and Proteomics Group, Australian Animal Health Laboratory, CSIRO Livestock Industries, Geelong VIC 3220, Australia b Department of Microbiology and Immunology, University of Melbourne, Parkville, VIC 3010, Australia ABSTRACT ARTICLE INFO Article history: Received 4 December 2007 Received in revised form 24 April 2008 Accepted 28 April 2008 Available online 14 May 2008 Keywords: Bacterial recovery Johne's disease Mycobacterium avium subspecies paratuberculosis Proteomic analysis Johne's disease is a slowly developing intestinal disease, primarily of ruminants, caused by Mycobacterium avium subspecies paratuberculosis. The disease contributes to significant economic losses worldwide in agricultural industry. Analysis of bacterial proteomes isolated directly from infected animals can provide important information about the repertoire of proteins present during infection and disease progression. In this study, M. avium subspecies paratuberculosis has been extracted from Johne's disease-infected cattle and goat intestinal tissue sections in a manner compatible with direct 2-DE proteomic analysis for comparison with in vitro-cultured bacteria. M. avium subspecies paratuberculosis was harvested from the submucosa and mucosa of intestinal sections and enriched from macerated tissue by hypotonic lysis, sonication and centrifugation through a viscosity gradient. Subsequent comparison of the proteomes of the in vivo- and in vitro-derived bacteria identified a number of proteins that were differentially expressed. Among them, a number of hypothetical proteins of unknown function and a hypothetical fatty acyl dehydrogenase (FadE3_2) and 3-hydroxyacyl-CoA dehydrogenase, possibly important for in vivo metabolism, utilising the pathway for the β-oxidation of fatty acids. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved. 1. Introduction There is a direct correlation between gene expression, protein synthesis and environmental stimuli to which bacteria are exposed. In a number of studies bacteria were cultured in vitro under varying conditions, including oxygen availability, heat stress, low pH were compared with those engulfed by macrophages to demonstrate the effect such environmental factors can have on protein and gene expression (Sturgill-Koszycki et al.,1996; Florczyk et al., 2001; Brunori et al., 2004). Within an infected host bacteria are likely to express a range of proteins important for pathogenicity, which may not be expressed under in vitro culture conditions. Therefore the comparison of the proteome of laboratory-cultured Mycobacterium avium sub- species paratuberculosis (M. paratuberculosis) with that of M. para- tuberculosis isolated from naturally-infected ruminants represents a means by which such potential virulence factors may be identified. To undertake such a comparison, target bacteria must be extracted and separated from eukaryotic cellular material, (i.e., from host tissue) and other co-existent microflora, with minimal modifications to the extracted bacteria. Johne's disease (JD) or paratuberculosis is a chronic intestinal wasting disease, primarily of cattle, goats, sheep and other ruminants, caused by the acid-fast bacterium M. paratuberculosis (Cocito et al., 1994). The disease manifests itself as a chronic granulomatous infection of the intestine, resulting in progressive weight loss, emaciation, cachexia, which can lead to animal death, and as such is of significance to agriculture production worldwide (Buergelt and Duncan, 1978; Benedictus et al., 1987; Clarke, 1997). During the development of JD, M. paratuberculosis resides within macrophages in infected gut tissue and also in the surrounding draining mesenteric lymph nodes (Momotani et al., 1988; Sigurdardottir et al., 2001). A characteristic feature of mycobacteria is the thick, waxy cell wall, a highly impermeable outer surface, which enables mycobacteria to survive in extreme environmental conditions and the presence of antibiotics (Rastogi, 1991; Jarlier and Nikaido, 1994; Brennan and Nikaido, 1995; Sung and Collins, 1998; Manning and Collins, 2001). The cell wall of mycobacteria also contributes to the survival of the mycobacteria within host macrophages, where the bacterium resides after initial infection of the Peyer's patches of the intestine and also in macrophages in the surrounding draining mesenteric lymph nodes (Momotani et al., 1988; Sigurdardottir et al., 2001; Tessema et al., 2001). This feature of mycobacterial cell wall can be exploited for the isolation and separation of mycobacteria from infected tissues, as Journal of Microbiological Methods 75 (2008) 29–39 Abbreviations: AFB, acid-fast bacilli; dpi, dots per inch; M. paratuberculosis, Myco- bacterium avium subspecies paratuberculosis; ZN, Ziehl Neelsen-staining. ⁎ Corresponding author. Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, Vic, 3220, Australia. Fax: +61 3 5227 5555. E-mail address: Wojtek.Michalski@csiro.au (W.P. Michalski). 1 These authors contributed equally to this work. 0167-7012/$ – see front matter. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2008.04.008 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth