Proteome and Differential Expression Analysis of Membrane and Cytosolic Proteins

2 Little is known of the protein expression in Mycobacterium avium subspecies paratuberculosis 3 (MAP) and how this contributes to pathogenesis. In the present study, proteins from both membranes 4 and cytosol were prepared from two strains of MAP; a laboratory-adapted strain K-10 and a recent 5 isolate, strain 187, obtained from a cow exhibiting clinical signs of Johnes disease. SDS-PAGE of 6 cytosol and membrane proteins from K-10 and 187 showed marked differences in protein expression. 7 Relative levels of protein expression from both MAP strains were measured using amine-reactive 8 isobaric tagging reagents (iTRAQ) and tandem mass spectroscopy. Protein identification and relative 9 expression data were obtained for 874 membrane and cytosolic proteins from the MAP proteome. 10 These data showed a number of significant differences in protein expression between K-10 and the 11 clinical isolate 187. Examples of proteins expressed at higher levels in strain 187 as compared to strain 12 K-10 are AtpC, RpoA and several proteins involved in fatty acid biosynthesis. In contrast, proteins 13 such as AhpC and several proteins involved in nitrogen metabolism were expressed at higher levels in 14 strain K-10 as compared to strain 187. These data may provide insights into the proteins whose 15 expression is important in natural infection but are modified once MAP is adapted to laboratory 16 cultivation. Results from these studies will provide tools for developing a better understanding of MAP 17 infection in the host, and offer potential as diagnostic reagents and vaccine candidates. 18 AC CE PT ED on S etem er 9, 2017 by gest http/jb.asm .rg/ D ow nladed fom Introduction 1 Paratuberculosis (Johne's disease) is a significant economic problem in cattle and sheep 2 worldwide (33). The causative agent of this chronic enteric disease is Mycobacterium avium subsp. 3 paratuberculosis (MAP). MAP is a gram positive intracellular pathogen that can persist and replicate 4 within macrophages of the infected host (41). It is thought that neonatal cattle become infected by 5 ingesting MAP shed in the feces of cattle that are in the clinical phase of the disease. Infections can 6 persist for several years in a subclinical phase that is difficult to identify due to the absence of clinical 7 signs. Once the disease enters the clinical phase, a thickening of the intestinal wall leads to weight loss, 8 decreased milk production, diarrhea, shedding of MAP in the feces, and eventual death (11, 16, 33). 9 Diagnosis of Johne’s disease is often complicated due to the slow progression of the disease and the 10 prevalence of genetically similar mycobacterial species in the environment. 11 The recent sequencing and annotation of the K-10 strain of MAP has led to significant progress 12 in the analysis of the genetic regulation of MAP (22). Microarray analysis has provided extensive data 13 on gene expression in several species of mycobacteria including MAP. Although single proteins have 14 been identified as potential diagnostic tools for MAP, comprehensive surveys of the MAP proteome 15 have been lacking until now. Recent studies have reported success in the use of methods such as 216 dimensional electrophoresis and surface enhanced laser desorption/ionization time of flight mass 17 spectrometry to profile protein expression in MAP (10, 15). Direct comparison of gene expression 18 between strains or within a single strain grown in differing conditions has proven useful in identifying 19 mycobacterial growth and pathogenicity characteristics (14, 17). Analysis of the MAP proteome offers 20 another level of regulation for study. 21 In post-genomic studies, high throughput HPLC coupled with MS has been used to identify 22 greater than 25% of the predicted M. tuberculosis coding sequences (25). The identification of large 23 sets of proteins like this is possible with extensive fractionation of the protein sample. Large-scale 24 proteome analysis from multiple samples has thus far been limited by an inability to quantify MS 25 results. Use of recently developed amine-labeled isobaric tags now allows for the analysis of relative 26 AC CE PT ED on S etem er 9, 2017 by gest http/jb.asm .rg/ D ow nladed fom protein expression levels between samples (1). Using this method, we analyzed the levels of protein 1 expression from two MAP strains. 2 Studies on MAP have shown that adaptation to lab growth conditions often is accompanied by 3 changes in phenotype (5, 38). It is unknown at the present time how changes in phenotype correspond 4 to protein expression profiles. The two strains in this study were chosen to evaluate effects of in vitro 5 propagation of MAP on protein expression. Comparing the proteome expression profiles of two MAP 6 strains of different passage number may help us elucidate the roles of key components involved in 7 MAP growth and pathogenesis. In the current study, our goal was to obtain a comprehensive survey of 8 the MAP proteome, and to demonstrate the differences in protein expression in two MAP strains, 9 multiple-passage laboratory-adapted strain K-10 and low passage strain 187 obtained from a cow with 10 clinical Johnes disease. 11