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Microbiology (2013), 159, 380–391 DOI 10.1099/mic.0.062737-0 Metabolic adaptation of Mycobacterium avium subsp. paratuberculosis to the gut environment Mathias Weigoldt,1 Jochen Meens,1 Franz-Christoph Bange,2 Andreas Pich,3 Gerald F. Gerlach134 and Ralph Goethe13 Correspondence Ralph Goethe [email protected] 1 Institute for Microbiology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Hannover, Germany 2 Department of Medical Microbiology and Hospital Epidemiology, Medical School Hannover, Hannover, Germany 3 Institute for Toxicology, Medical School Hannover, Hannover, Germany Received 3 August 2012 Revised 30 November 2012 Accepted 3 December 2012 Knowledge on the proteome level about the adaptation of pathogenic mycobacteria to the environment in their natural hosts is limited. Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease, a chronic and incurable granulomatous enteritis of ruminants, and has been suggested to be a putative aetiological agent of Crohn’s disease in humans. Using a comprehensive LC-MS-MS and 2D difference gel electrophoresis (DIGE) approach, we compared the protein profiles of clinical strains of MAP prepared from the gastrointestinal tract of diseased cows with the protein profiles of the same strains after they were grown in vitro. LC-MSMS analyses revealed that the principal enzymes for the central carbon metabolic pathways, including glycolysis, gluconeogenesis, the tricaboxylic acid cycle and the pentose phosphate pathway, were present under both conditions. Moreover, a broad spectrum of enzymes for boxidation of lipids, nine of which have been shown to be necessary for mycobacterial growth on cholesterol, were detected in vivo and in vitro. Using 2D-DIGE we found increased levels of several key enzymes that indicated adaptation of MAP to the host. Among these, FadE5, FadE25 and AdhB indicated that cholesterol is used as a carbon source in the bovine intestinal mucosa; the respiratory enzymes AtpA, NuoG and SdhA suggested increased respiration during infection. Furthermore higher levels of the pentose phosphate pathway enzymes Gnd2, Zwf and Tal as well as of KatG, SodA and GroEL indicated a vigorous stress response of MAP in vivo. In conclusion, our results provide novel insights into the metabolic adaptation of a pathogenic mycobacterium in its natural host. INTRODUCTION Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease (paratuberculosis), a chronic granulomatous enteritis of ruminants (Manning & Collins, 2001; Harris & Barletta, 2001). Among the pathogenic mycobacteria, MAP exhibits a strong intestinal tissue tropism, as it preferentially infects and multiplies in 3These authors contributed equally to this paper. 4Present address: IVD GmbH, Heisterbergallee 12, 30453 Hannover, Germany. Abbreviations: BDC, bovine mucosa-derived cytoplasm; CDC, culturederived cytoplasm; COG, Clusters of Orthologous Groups of Proteins; DIGE, difference gel electrophoresis; MAP, Mycobacterium avium subsp. paratuberculosis; MTB, Mycobacterium tuberculosis; PPP, pentose phosphate pathway; TCA cycle, tricarboxylic acid cycle. Two supplementary figures and two supplementary tables are available with the online version of this paper. 380 the gut mucosa. This is of particular interest, since MAP has long been suggested to be associated with Crohn’s disease in humans (Greenstein, 2003). In cattle, MAP is transmitted primarily via the faecal–oral route to neonatal calves. During the subsequent preclinical phase (2–5 years), bacteria persist and multiply in subepithelial macrophages, causing a chronic inflammation (Clarke, 1997; Harris & Barletta, 2001). Pathological alterations are preferentially found in the distal jejunum, ileum and the ileocaecal junction (Clarke, 1997). In contrast to other mycobacterial diseases, granuloma formation at the site of infection is diffuse, resulting in a granulomatous enteritis with mucosal thickening. Furthermore, even at late stages of disease no caseous necrosis or ulceration occurs. Lesions in other areas are found less commonly, confirming the intestinal region as the major site of disease (Buergelt et al., 1978). The molecular mechanisms of MAP pathogenicity are still incompletely understood. Studies with macrophage cell Downloaded from www.microbiologyresearch.org by 062737 G 2013 SGM IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:20:55 Printed in Great Britain M. avium subsp. paratuberculosis proteome in the host lines or primary macrophage cells have confirmed that MAP shares many virulence mechanisms with Mycobacterium tuberculosis (MTB), particularly the ability to survive in the hostile environment of macrophages (Coussens, 2001). This indicates common mechanisms in the pathobiology of mycobacterial infections. In recent years, it has become clear that carbon metabolism is a major determinant of the pathogenicity of MTB. Analyses of MTB obtained from macrophages in vitro and from the lungs of mice and humans suggested that MTB adapts its central metabolism in vivo by utilizing hostderived lipids rather than glucose and glycerol, the primary carbon sources metabolized in vitro (Russell et al., 2010). MTB strains lacking the glyoxylate shunt or the gluconeogenic enzyme phosphoenolpyruvate carboxykinase are attenuated during the chronic phase of infection in a mouse model of pulmonary tuberculosis (Marrero et al., 2010; Muñoz-Elı́as & McKinney, 2005; McKinney et al., 2000). Furthermore, many gene expression analyses indicate that MTB uses various pathways that mediate oxidative stress resistance and cell wall synthesis to evade the host immune responses and adapt to its intracellular lifestyle in the macrophage (Mukhopadhyay et al., 2012). A better understanding of mycobacterial metabolism in the infected host is of critical importance for developing new anti-mycobacterial drugs and diagnostic tools. However, most, if not all pathomechanisms of MAP and other pathogenic mycobacteria have been deduced to a major extent from murine and other small-animal models or from in vitro systems. Little is known about mycobacterial protein expression in their natural hosts. To identify MAP factors expressed in the host, RNA- and protein-based approaches have been employed, although with limited outcomes, in order to characterize the metabolic situation of MAP in the host (Janagama et al., 2010; Egan et al., 2008; Hughes et al., 2007; Wu et al., 2007). Here we analysed cytosolic proteins of MAP isolated from the intestinal tissue of diseased cows and compared them with those of the respective mycobacteria grown in vitro. Our comprehensive proteome analyses enabled us to deduce adaptive changes of metabolic pathways of a pathogenic mycobacterium in its natural host at the proteome level. Adaptation to the host environment became apparent in specific metabolic aspects, including nutrient availability, responses to antimicrobial host reactions and increased need for energy. METHODS Mucosa- and culture-derived MAP. The two genetically different type II MAP strains (MW080610-2, here named strain 2; MW0808212, here named strain 3) have been described previously (Weigoldt et al., 2011). They were obtained from cows of different ages (3.6 and 7.9 years) with clinical signs of Johne’s disease. For protein preparation, the strains were grown in Middlebrook 7H9 broth or on Middlebrook 7H10 agar containing OADC (oleic acid, albumin http://mic.sgmjournals.org fraction V, glucose, catalase) and mycobactin J (2 mg l21) as described previously (Weigoldt et al., 2011). Preparation of cytoplasmic fractions. Preparation of cytoplasmic fractions of MAP from bovine gut mucosa and culture was performed as described previously (Weigoldt et al., 2011). Cytosolic proteins were concentrated by precipitation with TCA (15 %, v/v) and centrifugation at 12 000 g, 15 min, 4 uC. Pellets were washed twice with 80 % acetone and resuspended in standard cell lysis buffer [SCLB; 8 M urea, 2 M thiourea, 30 mM Tris/HCl, 4 % CHAPS (pH 8.5)]. Prior to 2D difference gel electrophoresis (2D-DIGE) and preparative 2D gel electrophoresis, protein samples were treated with the PlusOne 2-D Clean-Up kit (GE Healthcare) and resuspended in SCLB. Final protein concentrations were determined using the 2D Quant kit (GE Healthcare) and adjusted to protein concentrations of 10 mg ml21. DIGE minimal labelling and 2D gel electrophoresis. MAP obtained from three different affected intestinal locations for each cow and from three independent cultures of each of the two strains were used for the preparation of bovine mucosa-derived cytoplasm (BDC) and culture-derived cytoplasm (CDC), respectively. 2DDIGE experiments were performed with the three pairs of BDC– CDC preparations of each strain, using 50 mg of each preparation labelled with cyanine dye Cy3 or Cy5 (GE Healthcare). Minimal labelling was performed according to the manufacturer’s instructions. In parallel, a preparative 2D gel electrophoresis of the respective BDC was performed, loading 1 mg protein. An additional 2D-DIGE experiment was performed comparing CDC of the two distinct MAP strains. For 2D gel electrophoresis and 2D-DIGE, all samples were supplemented with an equal volume of rehydration buffer [7 M urea (Roth), 2 M thiourea (Sigma) and 4 % w/v CHAPS (Roth)] supplemented with 2 % of the respective IPG buffer and 2 % (v/v) DTT (GE Healthcare). Immobiline DryStrips pH 4–7, 24 cm (GE Healthcare), were rehydrated for 14–16 h using 450 ml rehydration buffer supplemented with 1 % of the respective IPG buffer and 2 % (v/v) DTT (GE Healthcare). Samples were loaded into anodal sample cups and focused as previously described (Weigoldt et al., 2011). Second-dimension gel electrophoresis was performed as previously described (Buettner et al., 2009). Preparative 2D electrophoresis gels were stained with colloidal Coomassie G-250, and prominent protein spots were excised and digested with trypsin as described previously (Weigoldt et al., 2011) before MALDI-TOF MS. Scanning of 2D gels and protein quantification. Differential 2D gels were scanned on a Typhoon Trio Scanner (GE Healthcare) at a resolution of 100 dots cm21 using filters with specific excitation and emission wavelengths for the Cy3 (580BP30; 532 nm and 580 nm) and Cy5 (670/BP30; 633 nm and 670 nm). Protein spot abundance was analysed by DeCyder version 6.0 (Differential Analysis Software, GE Healthcare) using the differential in-gel analysis module. Quantification was applied for filter-confirmed spots with slope .1.4, area ,420 and volume ,130 000. Proteins were considered as differentially expressed if an at least 1.5-fold expression difference was determined between BDC and CDC. Not all of the total of six 2DDIGE gels allowed the identification of all spots. Therefore, only those proteins were considered for Table 1 that could be clearly identified in at least one gel of the BDC–CDC preparation from each of the two strains and which showed an at least 1.5-fold expression difference on all gels where they could be identified. Protein identification by MALDI-TOF-MS. Dried samples were solubilized in 3 ml 50 % acetonitrile (ACN) and 0.1 % trifluoroacetic acid (TFA); 1 ml of the peptide solution was mixed with 1 ml a-cyano- 4-hydroxycinnamic acid (CHCA; 5 mg ml21), 50 % ACN, 0.1 % TFA and spotted on the target plate. Samples were analysed on a Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:20:55 381 Annotation Fatty acid and cholesterol metabolism MAP3716c MAP3115c MAP2101 MAP3399 MAP3694c MAP3861 MAP2436c MAP3392c MAP2698c MAP1302c MAP0595c Central metabolism MAP2670c MAP2671c MAP3478 MAP1177c MAP1688 MAP0827c MAP2276c Microbiology 159 MAP2593c Antimicrobial stress response MAP1668c MAP3525c MAP4265 MAP2411 MAP0187c MAP0467c Energy production MAP3207 MAP2453c MAP4227c MAP3698c H37Rv locus Rv0271c Putative function RCN* Molecular mass (kDa) pI COGD AccD5 FadE5 ScoB FadA4 FadE25_4 DesA2 BcpB 78.7 76.7 71.9 59.4 66.6 47.9 40.2 42.0 31.6 19.9 5.06 5.21 5.23 5.13 4.98 5.03 4.99 5.18 4.86 5.65 I I I I I I I I (2) O Putative acyl-CoA dehydrogenase Putative acyl-CoA dehydrogenase Putative acyl-CoA oxidase Propionyl-CoA carboxylase beta chain 2 Putative acyl-CoA dehydrogenase 3-Oxoacid CoA-transferase Acetyl-CoA acetyltransferase Putative acyl-CoA dehydrogenase Acyl-[acyl-carrier protein] desaturase BcpB (peroxiredoxin family). Bacterioferritin comigratory protein S-(Hydroxymethyl) glutathione dehydrogenase FadE6 FadE22 AdhB 40.2 5.07 C 6-Phosphogluconate dehydrogenase-like protein Glucose-6-phosphate 1-dehydrogenase Trehalose phosphatase Transaldolase Malyl-CoA lyase Citrate synthase 2-Oxoglutarate ferredoxin oxidoreductase subunit beta 1-Pyrroline-5-carboxylate dehydrogenase Gnd2 Zwf OtsB2 Tal CitA KorA 36.7 53.0 40.5 40.4 23.7 42.3 39.4 5.26 5.23 5.23 4.72 4.75 5.6 5.48 G G G G G C C RocA 59.1 5.77 C KatG FusA2 GroEL1 Rv3846 Rv3592 Catalase/peroxidase Elongation factor G Chaperonin GroEL1 Putative pyridoxamine 59-phosphate oxidase Putative superoxide dismutase Putative antibiotic biosynthesis monooxygenase 83.4 76.3 81.8 15.5 23.0 11.5 5.06 5.12 4.95 4.81 5.61 5.43 P J O (2) P R Rv3151 Rv1308 Rv3463 Rv0248c NADH dehydrogenase subunit G F0F1 ATP synthase subunit alpha Putative luciferase-like monooxygenase Succinate dehydrogenase iron–sulfur subunit 83.4 60.2 30.3 70.9 5.06 4.89 6.31 5.76 C C C C Rv3280 Rv0244c Rv2503c Rv1323 Rv3274c Rv1094 Rv1608c Rv0761c Rv1122 Rv1121 Rv3372 Rv1448c Rv0889c Rv2454c Rv1187 Rv1908c Rv0120c Rv3417c SodA NuoG AtpA SdhA *Reference Common Name. DFunctional classification of proteins has been adopted from the genome database of MAP K-10 (http://www.ncbi.nlm.nih.gov/sutils/coxik.cgi?gi=380) according to the COG database. Energy production and conversion (C), carbohydrate transport and metabolism (G), lipid transport and metabolism (I), translation (J), post-translational modification, protein turnover, chaperones (O), inorganic ion transport and metabolism (P), general function prediction only (R), not in COG (2). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:20:55 M. Weigoldt and others 382 Table 1. List and characteristics of proteins with increased expression in BDC M. avium subsp. paratuberculosis proteome in the host (a) (d) Not in COGs (–) Energy production and conversion (C) 116 Cell cycle control, mitosis and meiosis (D) Amino acid transport and metabolism (E) Nucleotide transport and metabolism (F) Carbohydrate transport and metabolism (G) Coenzyme transport and metabolism (H) 184 638 BDC strain 2 BDC strain 3 (b) Lipid transport and metabolism (I) 116 194 738 Translation (J) Transcription (K) (c) BDC strain 2 Replication, recombination and repair (L) CDC strain 3 CDC strain 2 BDC strain 3 CDC strain 2 53 57 15 41 Cell wall/membrane biogenesis (M) CDC strain 3 Posttranslational modification, protein turnover, chaperones (O) lnorganic ion transport and metabolism (P) Secondary metabolites biosynthesis, transport and catabolism (Q) General function prediction only (R) 99 Function unknown (S) 26 44 79 521 18 50 37 39 92 Signal transduction mechanisms (T) BDC (638) CDC (738) Intracellular trafficking and secretion (U) 0 15 2 4 6 8 10 12 Relative abundance (%) 14 16 Fig. 1. Distribution of proteins and functional classification identified by LC-MS-MS. MAP proteins identified in BDC preparations (a), CDC preparations (b) and core proteome present in all analysed samples (c). Relative abundance of MAP proteins identified in BDC preparations (black bars) and CDC preparations (white bars) assigned to COG (d). VoyagerDE Pro as described previously (Buettner et al., 2009) or an AB Sciex TOF/TOF 5800 mass spectrometer (both AB Sciex). For MALDI-TOF/TOF analysis, internal calibration on autolytic porcine trypsin peptides was applied for precursor MS spectra and external calibration with Glu-Fib fragments was used for MS-MS spectra. MS data were searched against the Swiss-Prot Database with carbamidomethylation of cysteines, oxidation of methionine and N-terminal acetylation as variable modification. A precursor mass deviation of 120 p.p.m. and 0.5 Da for MS-MS fragments was used. At least two peptides with a Mascot peptide ion score higher than 20 each or one peptide with a score higher than 55 were used as a threshold for protein identification. LC-MS-MS analysis. For this, 30 mg of BDC and CDC was separated by SDS-PAGE and stained with Coomassie brilliant blue. From each lane, seven gel slices were excised and trypsin digestion was performed as previously described (Weigoldt et al., 2011). Peptide extracts were combined, dried and redissolved in 10 ml 2 % ACN, 0.1 % TFA. LC-MS-MS analysis was performed on an LTQ Orbitrap Velos mass spectrometer (Thermo-Fisher Scientific) exactly as described recently (Böer et al., 2011). Data analysis was facilitated by proteome discoverer software 1.2 (Thermo-Fisher Scientific) and http://mic.sgmjournals.org the Mascot search algorithm. Mascot was set up to search a customized database generated using the UniProt database (release 2012_03). It includes MAP K10 (NCBI reference sequence: NC_002944.2; 4350 genes, 4323 protein entries in UniProt), and a total of 6760 reviewed bovine protein entries (searched for Bos taurus). A false discovery rate of 0.01 and a peptide-score of 30 were used. Proteins were stated to be identified if at least two peptides were detected. Data processing and bioinformatics. Verified datasets were organized according to their distribution in the database Clusters of Orthologous Groups of Proteins (COG). Pathway reconstruction was performed using the cellular overview tool from SRI’s pathway tools software (http://ecocyc.org/background.shtml) for proteins with a Reference Common Name (RCN). In order to obtain information possibly missed using the cellular overview tool, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was searched using MAP annotation numbers, and MTB homologues were identified by protein homology BLAST using the Multi-Genome Homology Comparison (Comparative Tools) of Comprehensive Microbial Resource (CMR) available at http://cmr.jcvi.org and the TB database (http://genome.tbdb.org). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:20:55 383 M. Weigoldt and others pI 4 pI 7 MW (KDa) 100 ClpB NuoG KatG FadE6 AtpA ArgH ArgG FadA4 GlnA1 FadE5 66 45 Pgk Tal RocA GlyA MAP2276c CitA FadE25_4 35 MdH MmaA2 Gap FadB4 MAP4227c MAP0494 25 FixA SodA 15 Fig. 2. Representative 2D-DIGE gel of cytoplasmic proteins of in vivo- and in vitro-grown MAP. The CDC sample was prepared from strain 3 and labelled with Cy3 (green); the corresponding BDC preparation from strain 3 was labelled with Cy5 (red). A set of 25 proteins identified by MALDI-TOF are indicated. For abbreviations, see Table S1. RESULTS Identification of proteins by LC-MS/MS analysis Using LC-MS-MS we compared the protein profiles of BDC of the two MAP strains with their respective CDC. In the BDC preparations, we identified 938 different MAP proteins, 638 of which were present in both strains (Fig. 1a, Table S1a, available with the online version of this paper). Despite intensive purification, LC-MS-MS revealed the presence of a considerable number of contaminating bovine proteins such as fibrillin, collagen, actin, fibrinogen and fibronectin (67 proteins in both samples; Table S1a). A total of 1048 different MAP proteins were identified in CDC preparations with 738 proteins present in both strains (Fig. 1b, Table S1b). Due to culturing in Middlebrook 7H9 broth containing OADC; BSA and transthyretin were detected in the CDC preparations in addition to MAP proteins. We found a similar distribution of proteins in BDC and CDC preparations in 18 different functional COG categories (Fig. 1d). To define the core proteome that is expressed both in vivo and in vitro, we compared the BDCand CDC-derived proteins and found 521 proteins present in all preparations (Fig. 1c, Table S1c). These proteins represented enzymes of the central carbon metabolism 384 [glycolysis, glyconeogenesis, pentose phosphate pathway (PPP) and tricarboxylic acid (TCA) cycle] (Fig. 3, Table S1). We found in vivo and in vitro expression of six of the 34 annotated FadD, 11 of the 43 annotated FadE, three of the four annotated FadB, and four of the 10 annotated FadA proteins of the fatty acid b-oxidation cycle as well as the protein components of the methyl-citrate cycle and the glyoxylate shunt (Table S1c). Moreover, we found that nine MAP orthologues of proteins which are necessary for MTB growth on cholesterol (Griffin et al., 2011) were expressed both in vivo and in vitro. 2D Gel electrophoresis and protein identification by MALDI-TOF-MS Proteins of CDC and BDC were further analysed by quantitative 2D-DIGE, spot isolation from the corresponding preparative Coomassie-stained 2D gels and MALDITOF-MS analysis. Since the two MAP strains used in this study represented different genotypes (Weigoldt et al., 2011), we initially compared CDC samples from both strains by 2DDIGE, which resulted in a highly similar protein pattern (Fig. S1a). Next, 37 identically located protein spots representatively picked from preparative 2D gels of BDC preparations of the two strains were identified as identical protein matches (Fig. S1b, c). These results confirmed the high similarity of both types of protein samples. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:20:55 Microbiology 159 M. avium subsp. paratuberculosis proteome in the host Furthermore, we performed six 2D-DIGE experiments comparing BDC versus CDC from the two strains. Spots of interest were picked and analysed by MALDI-TOF-MS. A representative 2D-DIGE experiment with 25 identified proteins is shown in Fig. 2. In total, we analysed 309 protein spots in the BDC and CDC of both strains, representing 153 different MAP and four different bovine proteins (Table S2). compartment (Kuehnel et al., 2001). This so-called ‘recycling endosome’ is segregated from the late endosomal network, but is still able to communicate with early endosomes by which mycobacteria may acquire nutrients from outside the cells (Russell et al., 2010). MAP survives in macrophages of the distal region of the small intestine, the major site of host nutrient absorption, and, thus, one might assume that in the host MAP has access to a complex array of nutrients. Quantification of protein expression by 2D-DIGE Quantitative proteomics studies are limited in that the enzymic activity, turnover rates and secondary protein modifications which might be important are missed by these methods. Nevertheless, currently, together with transcriptional profiling studies, they are the only means of measuring metabolic adaptation during infection of the natural host. Proteomic data from pathogenic mycobacteria in their natural hosts are scarce, because of the difficulties of obtaining bacterial material in sufficient amount and quality. Initial work comparing mucosa- and culture-derived whole-cell lysates of MAP was done by Hughes et al. (2007) and Egan et al. (2008). Both groups performed 2D gel electrophoresis and estimated differences in protein abundance by silver staining. However, both studies were limited due to the low number of proteins that could be identified and limitations of silver-stained gels for protein quantification. In order to avoid these technical limitations, we used LC-MS-MS and 2D gel electrophoresis/DIGE with subsequent MALDI-TOF-MS. These methods allow quantification of protein levels between different biological samples (Bell et al., 2012; Tonge et al., 2001). Furthermore, 2D-DIGE with subsequent MALDI-TOF-MS possesses high reproducibility between replicates, and small differences in protein levels are detectable (Friedman et al., 2007). Among the proteins identified using densitometric spot quantification in 2D-DIGE gels, we were able to identify 29 proteins with differential expression between BDC and CDC (1.5-fold or higher expression in BDC compared with CDC; Table 1, Fig. S2a–d) and 32 proteins with similar expression (Table 2). Among the 32 proteins with similar expression were five central enzymes necessary for glycolysis and gluconeogenesis (Pgi/MAP0891c, Gap/MAP1164, Pgk/MAP1165, Tpi/ MAP1166, MAP4308c/putative fructose-bisphosphate aldolase class I) and also five proteins of the canonical mycobacterial TCA cycle (GltA2/MAP0829, Icd1/MAP3455c, GabD1/MAP3673c, Mdh/MAP2541c and Fum/MAP2693). Similar expression levels were also found for SucC/MAP0896 and SucD/MAP0897, which can convert succinyl-CoA from the methylmalonyl pathway to succinate, and also for enzymes of the glyoxylate shunt (AceAb/MAP1643 and GlcB/ MAP1549c). The 29 differentially expressed proteins were grouped according to their putative roles in MAP metabolism in the gut (Table 1). Thus, 11 proteins (FadE6/MAP3716c, FadE22/MAP3115c, MAP2101, AccD5/MAP3399, FadE5/ MAP3694c, ScoB/MAP3861, FadA4/MAP2436c, FadE25_4/ MAP3392c, DesA2/MAP2698c, BcpB/MAP1302c and AdhB/ MAP0595c) were assigned to fatty acid and cholesterol metabolism (Fig. S2a). Eight proteins (Gnd2/MAP2670c, Zwf/MAP2671c, OtsB2/MAP3478, Tal/MAP1177c, MAP1688, CitA/MAP0827c, KorA/MAP2276c and RocA/MAP2593c) were assigned to central metabolism (Fig. S2b). The antimicrobial stress response was represented by six differentially expressed proteins (KatG/MAP1668c, FusA2/ MAP3525c, GroEL1/MAP4265, MAP2411, SodA/MAP0187c and MAP0467c) (Fig. S2c), and another four proteins (NuoG/ MAP3207, AtpA/MAP2453c, MAP4227c and SdhA/ MAP3698c) were required for energy production (Fig. S2d). DISCUSSION It is now widely accepted that pathogenic bacteria adapt their metabolism to the nutrient availability provided by respective host niches (Eisenreich et al., 2010; Muñoz-Elı́as & McKinney, 2006). Similar to MTB, in the host, MAP infects and multiplies in macrophages (Zurbrick & Czuprynski, 1987). MAP inhibits phagosomal maturation and, like MTB, it resides in a specialized phagosomal http://mic.sgmjournals.org The qualitative LC-MS-MS-identification of 521 proteins present in both BDC and CDC point to metabolic pathways that are active in MAP both in vitro and in vivo. We were able to detect all principal enzymes of the central carbon metabolism (Fig. 3). MAP also expressed all components for b-oxidation of lipids in vivo and in vitro. Accordingly we found expression of enzymes of the glyoxylate shunt (AceAb/MAP1643 and GlcB/MAP1549c). The simultaneous expression of isocitrate dehydrogenase (Icd1) indicates that the canonical TCA cycle is also active. Of the 29 proteins that showed higher expression levels in vivo, 11 represented enzymes of the b-oxidation pathway. MTB lacks a canonical a-ketoglutarate dehydrogenase (Rhee et al., 2011). Interestingly, the anaerobic type 2oxoglutarate ferredoxin oxidoreductase (MAP2276c) also showed a higher protein level in vivo. The homologue in MTB (KOR, Rv2454c) drives a variant of the canonical TCA cycle by forming succinyl-CoA from a-ketoglutarate. This atypical TCA cycle is driven by b-oxidation, while in the absence of b-oxidation, a-ketoglutarate is transformed via succinic semialdehyde to succinate by the enzymes of the a-ketoglutarate decarboxylase pathway, Kgd and Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:20:55 385 M. Weigoldt and others MAP 1557c/gnd MAp 2670c/gnd2 Ribulose 5phosphate MAP 3884/fgd MAP 2671c/zwf MAP 1176c/zwf2 6-Phosphogluconate MAP 1175c/opcA MAP 1174c/devB MAP 2285c/rpi MAP 1135 rpe Ribose 5phosphate Xylose 5phosphate Glycogen/glucan Glucose MAP 2432c glpG MAP 2819 MAP 3146c ppgK pgmA Glucose 1-phosphate Glucose 6-phosphate MAP 0891c MAP 0573c/otsA MAP 0924 pgi MAP 3478/otsB galU Fructose 6-phosphate MAP 1178c/tkt MAP 2692 MAP 3044c/pfkA Sedoheptulose 7phosphate Trehalose Fructose 1,6-bisphosphate MAP 4308c MAP 1177c/tal Fructose 6phosphate Erythrose 4phosphate Glyceraldehyde 3Dihydroxyacetone phosphate phosphate MAP 1166/tpiA MAP 1164/gap MAP 1178c/tkt 1,3-Bisphosphoglycerate MAP 1165/pgk 3-Phosphoglycerate MAP 3981/gpmA 2-Phosphoglycerate β Oxidation of MAP 0990/eno MAP 3646/pckG fatty acids MAP 1169/ppc Phosphoenolpyruvate MAP 1310/pykA MAP 2540c/mez PropionylCoA Pyruvate MAP 1994/ace; MAP 1956/sucB MAP 3956/lpdC Acetyl-CoA MAP 0294c pca MAP 0827c/citA; MAP 0829/gltA2 MAP 3404 accA3 MAP 3399 accD5 Methylcitrate MAP 1201 acn Oxaloacetate MAP 0297c prpD Citrate MAP 2310c citE MAP 2541c mdh MAP 1201c/acn Malate Isocitrate Methylisocitrate MAP 2693 fum MAP 1613 Fumarate aceAb S-Methymalonyl-CoA MAP 1549c/glcB MAP 1643 MAP 1688 Glyoxylate aceAb Succinate R-Methymalonyl-CoA Succinyl-CoA MAP 3673c gabD1 MAP 3861 scoB Acetoacetate Glutamate Succinic semialdehyde MAP 2536 kgd MAP 1041c gabT MAP 4257 gadB GABA Acetoacetyl-CoA GabD1/D2 (Baughn et al., 2009). The increased expression of the oxoacid-CoA transferase ScoB (MAP3861), which catalyses the reversible conversion of 3-oxoacid-CoA and succinate to succinyl-CoA, indicates also that lipids are favoured as carbon source in vivo. 386 MAP 2593c rocA MAP 2276c korA MAP 0896/sucC MAP 0897/sucD Succinyl-CoA MAP 1225/mutA MAP 1226/mutB MAP 3455c/icd1 MAP 3455c/icd2 α-Ketoglutarate MAP 3443/sdhA MAP 3444/sdhB Proline MAP 3991 proC 1-Pyrroline-5carboxylate Host cholesterol has been proposed as another mycobacterial carbon source in vivo. It has been shown that MTB relies on cholesterol degradation for full virulence (Chang et al., 2009; Griffin et al., 2011; Nesbitt et al., 2010; Pandey & Sassetti, 2008; Sassetti & Rubin, 2003; Van der Geize Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:20:55 Microbiology 159 M. avium subsp. paratuberculosis proteome in the host Fig. 3. Scheme of the metabolic pathway network of M. avium subsp. paratuberculosis (adopted from Rhee et al., 2011; Baughn et al., 2009). Overview of proteins belonging to the major metabolic pathways identified in this study. MAP locus tags and gene abbreviations of proteins detected by LC-MS/MS in BDC and CDC samples of both strains are indicated. Red type indicates proteins with higher expression in vivo as determined by 2D-DIGE and MALDI-TOF-MS experiments. aceAb, isocitrate/methylisocitrate lyase; accA3, accD5, propionyl CoA carboxylase; aceE, dlaT and lpdC, pyruvate dehydrogenase E1 and E2 components; acn, aconitase/methylaconitase; citA, citrate synthase 2; citE, citrate lyase; CoA, coenzyme A; devB, 6phosphogluconolactonase; eno, enolase; fgd, F420-dependent glucose-6-phosphate dehydrogenase; fructose 1,6bisphosphatase; fum, fumarate hydratase; gabD1, succinic semialdehyde dehydrogenase, gabT, GABA transaminase; gadB, glutamate decarboxylase; galU, glucose-1-phosphate uridylyltransferase; gap, glyceraldehyde-3-phosphate dehydrogenase; glcB, malate synthase, glgP, glycogen phosphorylase; glpX, fructose 1,6-bisphosphatase; gltA2, type II citrate synthase; gnd and gnd2, 6-phosphogluconate dehydrogenase; icd1 and icd2, isocitrate dehydrogenase; kgd, a-ketoglutarate decarboxylase; kor, a-ketoglutarate ferredoxin oxidoreductase; methylmalonyl CoA epimerase; mdh, malate dehydrogenase, mutAB, methylmalonyl-CoA mutase, small and large subunit, mez, malic enzyme; opcA, glucose-6-phosphate dehydrogenase assembly protein; otsA, trehalose-phosphate synthase; otsB, trehalose-phosphate phosphatase; ppgK, glucokinase; pca, pyruvate carboxylase; pckA, phosphoenolpyruvate carboxykinase; pfkA, phosphofructose kinase; pgi, phosphoglucose isomerase; pgk, phosphoglycerate kinase; pgmA and gpmA, phosphoglycerate mutase; ppc, phosphoenolpyruvate carboxylase; proC, pyrroline 5-carboxylate reductase; prpD, methylcitrate dehydratase; pykA, pyruvate kinase; rocA, 1pyrroline-5-carboxylate dehydrogenase; rpe, ribulose-phosphate 3-epimerase; rpi, ribose-5-phosphate isomerase; scoB, 3oxoacid CoA transferase, sdhAB, succinate dehydrogenase flavoprotein subunits; sucCD, succinyl-CoA synthetase beta and alpha subunits; tal, transaldolase; tkt, transketolase; tpiA, triosephosphate isomerase; zwf and zwf2, glucose-6-phosphate 1dehydrogenase; GABA, gamma-aminobutyrate. et al., 2007). Among the nine MAP orthologues of proteins that have been described to be necessary for mycobacterial growth on cholesterol and which were expressed in vivo and in vitro, HsaA/MAP0497 and HsaD/MAP0498 are involved in cholesterol ring degradation, and FadD36/ MAP2580c and FadE25/MAP3392c are involved in sidechain degradation. The constitutive expression of these proteins even in the absence of cholesterol (in vitro) suggests metabolic ‘preparedness’ of MAP for different carbon sources. Several enzymes of cholesterol metabolism displayed increased protein levels in MAP in vivo, such as AccD5, the propionyl-CoA carboxylase beta chain. Propionyl-CoA carboxylase converts propionyl-CoA, derived from the degradation of odd-numbered fatty acids or the cholesterol side chains, into methylmalonyl-CoA. Methylmalonyl-CoA is further converted to succinyl-CoA, which can enter the TCA cycle. The likewise-upregulated enzymes acyl-CoA dehydrogenase FadE5 and acetyl-CoA acetyltransferase FadE25 have been reported recently to be required for MTB growth on cholesterol in vitro (Griffin et al., 2011). In addition, we observed an increased abundance of AdhB and BcpB, whose orthologues in MTB were identified to be also essential for growth on cholesterol (Griffin et al., 2011). Support for the hypothesis of MAP feeding on cholesterol inside the host was also provided by increased expression of an orthologue of the MTB MCE-family protein Mce4D (MAP0567/Rv3496c), a cholesterol transport protein identified in the membrane fraction of mucosa-derived MAP in our previous study (Weigoldt et al., 2011). In the host, MAP carbon metabolism seems to rely on the TCA cycle. This is further supported by the enhanced expression of RocA and of CitA. RocA is encoded on a predicted operon together with a proline dehydrogenase (MAP2592c). Both enzymes are found in MTB (Rv1187–Rv1188) and are http://mic.sgmjournals.org responsible for the two-step degradation of proline to glutamate. Glutamate, after conversion to 2-oxoglutarate or via the GABA shunt, can enter the TCA cycle. Since the genes for the generation of proline from arginine are not present in MAP, the increased expression of RocA in MAP in the host might indicate that MAP increasingly metabolizes proline provided from the host. One source might be host collagen. This could reflect a special adaptation of MAP grown in the ruminant host, since proline can accumulate in plants (Verbruggen & Hermans, 2008) and might be provided in high amounts through the diet of ruminants. We identified four enzymes of the PPP with higher expression in BDC. Zwf (MAP2671c) and Gnd2 (MAP2670c) encode a glucose-6-phosphate 1-dehydrogenase and a 6-phosphogluconate dehydrogenase, respectively, which catalyse the conversion of glucose 6-phosphate to ribulose 5-phosphate. Tal (MAP1177c) is a transaldolase required for the production of fructose 6-phosphate and erythrose 4-phosphate in the non-oxidative branch of the PPP. The mycobacterial PPP is necessary for the generation of NADPH, which serves as reducing agent in many biosynthetic pathways as well as in the oxidative stress response, and as a pentose source for nucleotide biosynthesis and for arabinose, which is an essential part of the mycobacterial cell envelope (Alderwick et al., 2011). Thus, increased expression of PPP in vivo might serve various purposes in adaptation of MAP during infection. After crossing the intestinal barrier, MAP infects and survives inside macrophages, where it has to resist reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Ehrt & Schnappinger, 2009). Among the more highly expressed MAP proteins necessary for combating stress, SodA converts superoxide radicals to hydrogen peroxide, which is converted into water and oxygen by KatG. An upregulation of katG in host-derived MAP at the mRNA Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:20:55 387 Annotation Glycolysis/gluconeogenesis MAP0891c MAP1164 MAP1165 MAP1166 MAP3044c MAP3146c MAP3981 MAP4308c PPP MAP1557c TCA cycle MAP0829 MAP0896 MAP0897 MAP1549c MAP1643 MAP2541c MAP2693 MAP3455c MAP3673c Amino acid transport and metabolism MAP1024 MAP1361 MAP1365 MAP1367 MAP1368 MAP1962 MAP2661 MAP2699c H37Rv locus Putative function RCN* Molecular mass (kDa) pI COGD Rv0946c Rv1436 Rv1437 Rv1438 Rv3010c Rv3068c Rv0489 Glucose-6-phosphate isomerase Glyceraldehyde-3-phosphate dehydrogenase Phosphoglycerate kinase Triosephosphate isomerase 6-Phosphofructokinase Phosphoglucomutase Phosphoglyceromutase Fructose-1,6-bisphosphate aldolase Pgi Gap Pgk Tpi PfkA PgmA Gpm 60.7 36.1 42.4 27.5 37.0 58.0 27.2 33.6 5.32 5.22 4.84 5.23 6.06 5.48 5.42 5.31 G G G G G G G G Rv1844c 6-Phosphogluconate dehydrogenase Gnd 52.8 5.23 G Rv0896 Rv0951 Rv0952 Rv1837c Rv1916 Rv1240 Rv1098c Rv3339c Rv0234c Type II citrate synthase Succinyl-CoA synthetase subunit beta Succinyl-CoA synthetase subunit alpha Malate synthase G Isocitrate lyase Malate dehydrogenase Fumarate hydratase Isocitrate dehydrogenase 1 Succinic semialdehyde dehydrogenase GltA2 SucC SucD GlcB AceAb Mdh Fum Icd1 GabD1 48.3 43.2 30.8 80.3 85.2 34.6 49.7 46.4 50.0 5.09 4.95 5.23 4.83 5.35 4.87 5.36 5.63 4.77 C C C C C C C C C Rv1077 Rv1652 Rv1656 Rv1658 Rv1659 Rv2220 Rv1133c Putative cystathionine beta-synthase N-Acetyl-gamma-glutamyl-phosphate reductase Ornithine carbamoyltransferase Argininosuccinate synthase Argininosuccinate lyase Glutamine synthetase 5-Methyltetrahydropteroyltriglutamate—homocysteine methyltransferase Serine hydroxymethyltransferase CysM2 ArgC ArgF ArgG ArgH GlnA1 MetE 49.2 35.3 33.6 43.8 49.7 53.7 81.6 5.1 5.86 4.9 5.21 5.06 4.96 5.02 E E E E E E E GlyA 45.0 5.75 E Rv1093 Microbiology 159 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:20:55 M. Weigoldt and others 388 Table 2. List and characteristics of proteins of the major metabolic pathways with similar expression in BDC and CDC Rv0859 Rv0215c Rv0644c MAP0789 MAP3651c MAP4095c http://mic.sgmjournals.org *Reference Common Name. DFunctional classification of proteins has been adopted from the genome database of MAP K-10 (http://www.ncbi.nlm.nih.gov/sutils/coxik.cgi?gi=380) according to the COG database. Energy production and conversion (C), amino acid transport and metabolism (E), carbohydrate transport and metabolism (G), lipid transport and metabolism (I), cell wall/membrane biogenesis (M). I I M 42.6 44.1 33.1 FadA_1 FadE3_2 MmaA2 5.03 6.15 5.12 I I I 5.18 12.0 5.15 30.8 46.1 40.8 FadB2 EchA3 GcpE 3-Hydroxybutyryl-CoA dehydrogenase Putative enoyl-CoA hydratase/isomerase (MAPs_39340) 4-Hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG) Acetyl-CoA acetyltransferase Putative acyl-CoA dehydrogenase Cyclopropane-fatty-acyl-phospholipid synthase Rv0468 Rv0632c Rv2868c Fatty acid transport and metabolism MAP3962 MAP4102c MAP2938c Annotation Table 2. cont. H37Rv locus Putative function RCN* Molecular mass (kDa) pI COGD M. avium subsp. paratuberculosis proteome in the host level has been shown using real-time PCR (Granger et al., 2004). The induction of both sodA and katG has been shown in culture at the transcriptional level in MTB after applying oxidative and nitrosative stress (Voskuil et al., 2011). Furthermore, expression of these proteins sustained the viability of MTB in mouse infection models (Edwards et al., 2001; Li et al., 1998). GroEL1 (MAP4265) protein levels were increased up to 21-fold in vivo. GroEL1 has high similarity to its counterpart in MTB (Rv3417c). In MTB, both GroEL1 and GroEL2 proteins are upregulated during heat shock (Stewart et al., 2002), the oxidative stress response (Dosanjh et al., 2005), and oxidative damage upon macrophage infection (Monahan et al., 2001). Moreover, some studies suggest their involvement in the immune response to MTB infection (Lewthwaite et al., 2001; Orme et al., 1993). Recently it has been reported that GroEL1 binds strongly to DNA and effectively functions as a DNA-protecting agent against DNase I or hydroxyl radicals (Basu et al., 2009). In addition, the orthologues of FusA2 and BcpB as well as the monooxygenase MAP0467c have been suggested to be involved in counteracting stress responses of MTB and MAP (Seshadri et al., 2009; Berney & Cook, 2010; Janagama et al., 2010; Jaeger, 2007). It seems that in the host, MAP exhibits enhanced metabolic activity. In agreement with this, expression of three enzymes involved in oxidative phosphorylation was strongly increased. NuoG belongs to the NADH : ubiquinone oxidoreductase complex I, the first component of the respiratory chain (Friedrich & Böttcher, 2004). MAP3698c is highly homologous to MTB Rv0248c a putative subunit of the succinate dehydrogenase (SdhA) of the respiratory chain. Lastly, MAP2453c/AtpA is a subunit alpha of the F0F1 ATPsynthase, which generates ATP within aerobic respiration. Overall, these data indicate that MAP, in the host, displays enhanced respiration, which is a further indication of MAP adaptation to the host environment. Conclusion Within the host, changes in MAP metabolism seem to be dominated by an adaptation to antimicrobial host reactions, which is indicated by the enhanced expression of protective enzymes such as SodA and KatG. Energy losses due to these processes might be compensated by an enhanced activity of the PPP and enhanced ATP generation through respiratory phosphorylation. The central metabolism appears to be driven by b-oxidation of alternative lipid sources, most probably cholesterol, which is abundant in host cell membranes. The acetyl-CoA generated enters an alternative TCA pathway in which a type 2 oxoglutarate ferredoxin oxidoreductase contributes to the conversion of a-ketoglutarate to succinyl-CoA. Upon growth on the TCA cycle, MAP uses the glyoxylate shunt. Carbon efflux for gluconeogenesis via phosphoenolypyruvate and for amino acid synthesis might be compensated by the generation of a-ketoglutarate from glutamate via degradation of proline. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 29 Apr 2017 20:20:55 389 M. Weigoldt and others Overall, our results provide novel insights into the metabolic adaptation of a pathogenic mycobacterium in its natural host. Edwards, K. M., Cynamon, M. H., Voladri, R. K., Hager, C. C., DeStefano, M. S., Tham, K. T., Lakey, D. L., Bochan, M. R. & Kernodle, D. S. (2001). Iron-cofactored superoxide dismutase inhibits host responses to Mycobacterium tuberculosis. Am J Respir Crit Care Med 164, 2213–2219. ACKNOWLEDGEMENTS Egan, S., Lanigan, M., Shiell, B., Beddome, G., Stewart, D., Vaughan, J. & Michalski, W. P. (2008). The recovery of Mycobacterium avium We are grateful to Klaus Doll (Clinic for Ruminants and Swine, Justus-Liebig-University, Giessen, Germany) for supporting the organization of infected cattle, and to Peter Valentin-Weigand for critical reading of the manuscript. This work was supported by the German Ministry for Science and Education (BMBF; ZooMAP, 01KI0750 and ZooMAPII 01KI1003A, 01KI1003B). R. G. was additionally supported by a grant from the German Research Foundation (DFG; Ge522/6-1). subspecies paratuberculosis from the intestine of infected ruminants for proteomic evaluation. 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