Department of Infectious Diseases
Antigen expression and metabolism of Mycobacterium avium subsp. paratuberculosis in vivo
THESIS
submitted in partial fulfilment of the requirements for the degree
Doctor rerum naturalium (Dr. rer. nat.)
by
Mathias Weigoldt from Freiberg, Saxony
Hannover 2012
Supervisor: Prof. Dr. Ralph Goethe
Supervision Group: Prof. Dr. Ralph Goethe Prof. Dr. Mathias Hornef Prof. Dr. Andreas Beineke
1st Evaluation: Prof. Dr. Ralph Goethe Institute for Microbiology
Department of Infectious Diseases
University of Veterinary Medicine Hannover
Prof. Dr. Andreas Beineke
Institute for Pathology
University of Veterinary Medicine Hannover
Prof. Dr. Mathias Hornef
Institute for Medical Microbiology and Hospital Epidemiology
Hannover Medical School
2nd Evaluation: Dr. Siegfried Weiß
Institute for Molecular Immunology
Helmholtz Centre for Infection Research, Braunschweig
Date of oral exam: 11.05.2012
This work was financially supported by the BMBF zoonosis network Mycobacterium avium ssp. paratuberculosis - from Johne’s Disease to Crohn’s Disease - (ZooMAP).
1. Chapter: General introduction... 9
1.1. Mycobacterium avium subsp. paratuberculosis and Johne’s disease... 9
1.2. Zoonotic potential of M. avium subsp. paratuberculosis... 10
1.3. Diagnosis of M. avium subsp. paratuberculosis infections ... 11
1.4. Virulence determinants of M. avium subsp. paratuberculosis and pathogenesis of Johne’s disease ... 11
1.5. Current knowledge of M. avium subsp. paratuberculosis mRNA and protein expression in the host... 12
1.6. Metabolic host adaptation of pathogenic mycobacteria ... 13
1.7. Challenges in research of the M. avium subsp. paratuberculosis pathogenesis ... 14
1.7.1 Experimental challenges in molecular biological analysis of M. avium subsp. paratuberculosis... 14
1.7.2 Sequencing and bioinformatics analysis ... 14
1.8. Aim of this study... 15
2. Chapter: Material and methods ... 25
2.1. Origin of animals and culture of M. avium subsp. paratuberculosis... 25
2.2. Strain typing of bovine isolates of M. avium subsp. paratuberculosis... 26
2.3. Isolation of M. avium subsp. paratuberculosis from surface-layer cultures or directly from mucosa ... 27
2.4. Cell disruption, protein preparation of membrane-enriched and cytoplasmic fractions from M. avium subsp. paratuberculosis... 29
2.5. 2D difference gel electrophoresis (2D DIGE) ... 30
2.6. Scanning, data analysis and protein identification by MALDI-TOF-MS ... 31
2.7. Tube-gel trypsin digestion ... 32
2.8. SDS-PAGE-based trypsin digestion ... 32
2.9. Nanoflow-liquid-chromatography-coupled tandem mass spectrometry (nUPLC- ESI Q-TOF-MS/MS) ... 34
2.10. High throughput protein analysis using nanoflow-liquid-chromatography- coupled tandem mass spectrometry (LC-orbitrap-MS/MS) ... 34
3. Chapter: Differential proteome analysis of Mycobacterium avium subsp.
paratuberculosis grown in vitro and isolated from cases of clinical
Johne’s disease ... 39
4. Chapter: Proteome profiling of Mycobacterium avium subsp. paratuberculosis isolates obtained from cows with clinical Johne’s disease reveals metabolic adaptation in the natural host ... 43
5. Chapter: General discussion... 103
5.1. Technical issues and experimental design ... 103
5.1.1 Challenges of providing protein samples from mucosa- and culture derived M. avium subsp. paratuberculosis... 103
5.1.2 Preparation of M. avium subsp. paratuberculosis from intestinal mucosa of cows with clinical symptoms of Johne’s disease ... 103
5.1.3 In vitro cultures of M. avium subsp. paratuberculosis... 104
5.1.4 Isolation of proteins from M. avium subsp. paratuberculosis... 105
5.1.5 Protein analysis and quantification... 105
5.2. Antigen expression of M. avium subsp. paratuberculosis isolated from intestinal mucosa of naturally infected cows... 107
5.2.1 Analysis of differential protein expression in the membrane fraction ... 107
5.3. Metabolism of M. avium subsp. paratuberculosis during clinical phase of JD... 109
5.3.1 Analysis of differential protein expression in the cytosolic fraction... 109
5.3.2 Metabolomic analysis of differentially expressed proteins... 110
5.4. Adaptation to host-defence induced stress... 111
5.5. Conclusions and Outlook... 112
6. Chapter: Summary... 121
7. Chapter: Zusammenfassung ... 125
8. Literature ... 129
9. Appendix ... 143
9.1. List of abbreviations ... 143
9.2. Complete results of 2D GE/2D DIGE: ... 146
9.3. Figures ... 171
9.4. Supplementary figures ... 171
9.5. Tables... 172
9.6. Supplementary tables... 172
9.7. Complete list of own publications: ... 174
Chapter 1
General introduction
1. Chapter: General introduction
1.1. Mycobacterium avium subsp. paratuberculosis and Johne’s disease
Mycobacterium avium subsp. paratuberculosis (MAP) is an acid-fast, rod-shaped bacterium belonging to the family Mycobacteriaceae within the phylum Actinobacteria subdivided into the order Actinomycetales (Rastogi et al., 2001; Ventura et al., 2007). The genera Mycobacterium, Corynebacterium and Nocardia are characterized by the presence of a waxy cell envelope containing mycolic acids, responsible for their acid-fast staining properties and high resistance against harsh environmental conditions and mechanical treatment which distinguish them from other bacteria (Rastogi et al., 2001). The mycobacterial cell envelope contains a large variety of lipids, branched mycolic acids which are covalently bound to the peptidoglycan of the cell wall forming an outer membrane-like layer (Daffe & Draper, 1998).
The family Mycobacteriaceae contains fast- and slow-growing species (Wayne & Kubica, 1986). Among them, the non pathogenic species Mycobacterium smegmatis grows fast in laboratory culture media and is therefore suitable to study molecular and metabolic features (Bardarov et al., 2002; Berney & Cook, 2010). In contrast to this species, MAP is one of the slowest growing mycobacteria (Lambrecht et al., 1988) and difficult to modify genetically, a fact which severely hampers research in the field of pathogenesis and metabolism. Other slow-growing species include Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium leprae, which are important animal and human pathogens. Other mycobacteria, genetically more closely related to MAP, are Mycobacterium avium subsp.
avium (MAA) and Mycobacterium avium subsp. hominissuis (MAH) two subspecies of M.
avium subclassified into the Mycobacterium avium complex (MAC) (Turenne et al., 2007).
MAA is the causative agent of tuberculosis in birds, in which it acts as an obligate pathogen (Thorel et al., 1997). MAH is more frequently isolated from pigs and responsible for lesions in lymph nodes of the digestive tract. Both MAC members are opportunistic pathogens that may infect several species, including humans and pigs (Agdestein et al., 2012). Despite the taxonomic relationship, MAA and MAP are phenotypically diverse organisms (Turenne et al., 2008).
MAP is the causative agent of Johne’s disease (JD) also known as paratuberculosis in ruminants. This disease, initially reported by H. A. Johne and L. Frontingham in 1895, has been characterized by a progressive wasting of animals with clinical signs of a granulomatous
Chapter 1 - General introduction
10
inflammation affecting the ileum. JD occurs worldwide (Manning & Collins, 2001) and most commonly affects domestic ruminants, particularly dairy cattle (Godfroid et al., 2000; Greig et al., 1999). The disease is untreatable and eventually results in death (Clarke, 1997).
Because of loss of milk production and early culling of cows, JD imposes a serious economic threat to the beef and dairy industry worldwide (Harris & Barletta, 2001; Hutchinson, 1996).
1.2. Zoonotic potential of M. avium subsp. paratuberculosis
In humans, the prevalence of MAC disease has increased dramatically in immunocompromised patients such as AIDS patients as well as the immunocompetent patient population (Collins, 1988; Collins, 1989; Prince et al., 1989). Ten years ago, MAP has been detected in a HIV-infected patient for the first time, thus suggesting a pathogenic role of MAP for immunocompromised patients (Richter et al., 2002).
On the other hand, the zoonotic potential such as an association of MAP with Crohn's disease (CD) has been discussed controversially (Abubakar et al., 2008; Rosenfeld & Bressler, 2010).
This discussion was initiated by a clinical and pathological resemblance between two chronic inflammatory bowel diseases, JD in cattle and CD in humans (Dalziel, 1989). CD is a chronic inflammatory bowel disease in humans with a mostly unknown aetiology. The incidence in the human population used to be low, but starting in the mid 1940s the incidence continued to climb in Western Europe and North America (Loftus, Jr. et al., 1998; Loftus, Jr. & Sandborn, 2002). At present, CD in Europe has an incidence of 9.8/100,000 (Loftus, Jr., 2004) and is considered to be a major human healthcare problem with high socio-economic importance in developed societies (Bodger, 2002). In addition to CD an association of MAP with type 1 diabetes mellitus (Rosu et al., 2009) and multiple sclerosis (Cossu et al., 2011) is discussed.
The source from which human patients acquire MAP is still unknown. Since MAP is not able to multiply in the environment, the primary source of human MAP infection must be assumed to be the infected animal. Milk contaminated with MAP could be a potential route of transmission to humans (Grant et al., 2002). This has initiated an ongoing controversial discussion on the number of MAP in milk and MAP from contaminated milk as a possible cause of CD (Grant, 2005). Other potential sources are water and meat; however, evidence supporting the consumption of foods containing MAP as a cause of CD is lacking (Abubakar et al., 2007; Rosenfeld & Bressler, 2010).
1.3. Diagnosis of M. avium subsp. paratuberculosis infections
The pre-clinical period of JD can last for several years with infected animals not showing clinical signs of disease and, therefore, infected animals are inconspicuous to the farmer.
Since MAP is transmitted to uninfected herds by these subclinically infected animals there is a need for tests facilitating early detection of carrier animals (Whitlock & Buergelt, 1996).
The tests currently in use either have a low diagnostic sensitivity and, therefore, are only applicable for herd-diagnosis (ELISA tests) or they are not sufficiently specific to find acceptance with the farmer (interferon-gamma test). Bacteriological culture - although having an analytical sensitivity of 10 colony forming units per gram of feces - also lacks diagnostic sensitivity due to intermittent shedding; in addition, it takes 6 to 8 weeks to grow the organism. Finally, the gold standard procedure - culture of the ileocecal lymph node (Merkal et al., 1987) - is not practical as a routine approach.
One way to improve immune response-based diagnostics would be the identification of novel antigens inducing an earlier (ELISA) or more specific response (interferon-gamma test) (Mikkelsen et al., 2011a; Mikkelsen et al., 2011b).
1.4. Virulence determinants of M. avium subsp. paratuberculosis and pathogenesis of Johne’s disease
In cattle, MAP is transmitted primarily by contaminated feaces or milk via the oral route to neonatal calves (Harris & Barletta, 2001; Taylor et al., 1981). Once within the intestinal lumen, MAP has the ability to penetrate the intestinal epithelial barrier and to enter the subepithelial lamina propria causing an invasive infection (Pott et al., 2009). Pathogenic mycobacteria express surface-exposed adhesion molecules such as heparin-binding hemagglutinin (HBHA) and laminin-binding protein (HupB) which promote binding to epithelial cells (de Lima et al., 2009; Vidal Pessolani et al., 2003). For MAP it was shown that preexposure to milk or to a hyperosmolar environment and intracellular passage in bovine mammary epithelial cells caused an increased efficiency of epithelial invasion (Patel et al., 2006). Interestingly, the surface exposed major membrane protein I (MMP-I/MAP2121c) exhibits a higher level of expression in low-oxygen and high-osmolarity conditions similar to the environment of the intestine and plays a role in invasion of bovine epithelial cells (Bannantine et al., 2003). Protein homology prediction using NCBI-BLAST search tool
Chapter 1 - General introduction
12
reveals this protein to be highly orthologous to MMP-I of M. leprae and MAC species, but not to M. tuberculosis, which, in turn, is primarily transmitted by the aerosol route.
After crossing the intestinal mucosa using Peyer’s patches and enterocytes MAP reaches the mesenteric lymph nodes possibly via dendritic cells (Bermudez et al., 2010; Wu et al., 2007a).
Experiments with macrophage cell lines or primary macrophage cells confirmed that MAP shares many virulence mechanisms with M. tuberculosis, particularly the ability to survive in the hostile environment of macrophages (Coussens, 2001; Kuehnel et al., 2001; Stabel, 2006).
One important mechanism for the intracellular survive in macrophages is the inhibition of phagosomal maturation (Hostetter et al., 2003; Kuehnel et al., 2001) and, most likely similar to M. tuberculosis, MAP also resides in a specialized phagosomal compartment, the so called
“recycling endosome”, which is segregated from the late endosomal network but still able to communicate with early endosomes and thus acquiring nutrients from the outside of the cells (Kuehnel et al., 2001; Russell, 2007). MAP is also able to activate macrophages to produce proinflammatory cytokines, but in contrast to MAA, MAP inhibits the antigen-specific stimulatory capacity of macrophages for antigen-specific CD4+ T cells (Zur Lage et al., 2003). Although MAP seems to share common general mechanisms in the pathobiology of mycobacterial infections, MAP is multiplying in macrophages in the small intestine which is a completely different ecological niche compared to M. tuberculosis which affects the lung of its human host where it is found within macrophages in distinct granulomas in a dormant or latent state remaining viable for many years without the patient showing clinical signs of tuberculosis (Wayne, 1994). Until now, however, little is known about the interplay between of virulence determinants of MAP and the host (Shin et al., 2006).
1.5. Current knowledge of M. avium subsp. paratuberculosis mRNA and protein expression in the host
The first report about differential gene expression comparing transcription levels between host- and culture-derived MAP mRNA revealed an upregulation of catalase/peroxidase katG as investigated by real time PCR (Granger et al., 2004). Three years later, first efforts to profile gene transcripts of MAP exposed to several stressors or isolated directly from infected cows revealed differential expression of genes involved in the universal stress response like heat shock proteins (e.g., hsp and htpX). This finding demonstrated shared principals between
acid stress, oxidative stress, heat shock and faecal sample environment (Wu et al., 2007b).
More recently, transcriptional profiles of MAP obtained from ileum and mesenteric lymph nodes of naturally infected cows as well as from a bovine monocyte-derived macrophage infection model, have been characterised (Janagama et al., 2010b).
Only a few studies on MAP have investigated differential protein expression in culture models under conditions mimicking infection such as nitrosative or oxidative burst (Kawaji et al., 2010) as well as hypoxia and nutrient starvation (Gumber et al., 2009). A further method to induce a stress response in MAP was the exposure to heat which induced a partially similar activation of metabolic pathways as hypoxia and starvation (Gumber et al., 2009; Gumber &
Whittington, 2009). A previous report comparing membrane and cytoplasmic fractions of low and high passage number culture-grown MAP strains by high throughput mass spectrometrical analysis resulted in the detection of 874 proteins (20% of all open reading frames annotated) (Radosevich et al., 2007).
Only two studies on MAP have investigated differential protein expression within natural infected hosts with clinical signs of JD. Initially MAP whole cell lysates directly obtained from gut mucosa of ovine (Hughes et al., 2007) and bovine hosts (Egan et al., 2008) were analyzed by comparative 2D GE and subsequent densitometric quantification of silver stained protein spots. The differentially expressed proteins identified by Egan et al. (2008) and Hughes et al. (2008) using this approach were different, which has been explained by the differences between sheep strains (S strains) and cattle strains (C strains). Indeed, both strain types exhibit differences at genomic level (Marsh et al., 2006; Semret et al., 2006) and a different iron-sparing response possibly due to different iron storage mechanisms (Janagama et al., 2010a). An alternative explanation is that the analyses in both studies did not cover all proteins differentially expressed and, therefore, missed concordant information.
1.6. Metabolic host adaptation of pathogenic mycobacteria
It is now widely accepted that pathogenic bacteria adapt their metabolism to the nutrient availability provided by a respective host niche for survival and growth (Eisenreich et al., 2010). As stated above, MAP persists inside endosomes of magrophages. It is believed that mycobacterial energy conversion and biomass production relies on fatty acids rather than on carbohydrates (McKinney et al., 2000; Schnappinger et al., 2003; Stokes & Waddell, 2009;
Van der Geize et al., 2007). Hosts’ cholesterol is discussed to be one important fatty acid
Chapter 1 - General introduction
14
source of the M. tuberculosis diet. It was shown that M. tuberculosis can metabolise cholesterol in vitro, and several genes for cholesterol degradation are required for full virulence in host infection models (Chang et al., 2009; Griffin et al., 2011; Nesbitt et al., 2010; Pandey & Sassetti, 2008; Rosloniec et al., 2009; Sassetti & Rubin, 2003; Van der Geize R. et al., 2011; Van der Geize et al., 2007). Moreover, M. tuberculosis strains defective in enzymes of the pyruvate dehydrogenase complex, the glyoxylate shunt or the gluconeogenic enzyme phosphoenolpyruvate carboxykinase were attenuated during the chronic phase of infection in a mouse model of pulmonary TB (Marrero et al., 2010; McKinney et al., 2000;
Munoz-Elias & McKinney, 2005; Shi & Ehrt, 2006).
1.7. Challenges in research of the M. avium subsp. paratuberculosis pathogenesis
1.7.1 Experimental challenges in molecular biological analysis of M. avium subsp.
paratuberculosis
Despite its clinical and economical relevance the molecular mechanisms of MAP pathogenicity are still poorly understood. As stated above, MAP is characterized by extremely low multiplication rates (Lambrecht et al., 1988), and difficulties in genetic modification (McFadden, 1996) hamper the applicability of standard molecular biology research tools.
The availability and analysis of biomolecules (DNA, RNA, protein) is severely impaired by a highly robust mycobacterial cell envelope. Furthermore it has been reported that the correlation of protein and mRNA expression is poor (Brunori et al., 2004).
1.7.2 Sequencing and bioinformatics analysis
Although genome sequencing of a bovine MAP isolate (MAP K-10) was an important step forward in understanding the bacteria (Li et al., 2005) resequencing in 2010 revealed 90 single-nucleotide errors, 28 frameshift alterations and a 51-bp indel (Wynne et al., 2010).
Although the original sequences have been corrected by Wynne et al. (2010), until today only the erroneous and poorly annotated MAP K-10 sequence has been accessible for protein database search tools. Thus, 1019 MAP proteins are not functionally classified according to the Cluster of Orthologous Groups (COG). In addition, 251 proteins are in group S (unknown function) and 621 proteins are associated with poorly described cellular pathways. The relatively poor annotation of MAP proteins is exemplary documented by the fact that 184 of
394 proteins functional categorised into COG I “lipid transport and metabolism” are assigned as hypothetical proteins. Furthermore, genomic sequence information from different MAP strains such as sheep strains is incomplete in the databases.
1.8. Aim of this study
Bovine Johne’s disease (JD) caused by MAP, poses a significant economic problem to the beef and dairy industry worldwide. Until now, pathogenesis of JD and the metabolic activity of MAP within the host are only partially resolved. Therefore, the aim of this study was the comprehensive analysis of the MAP proteome isolated from intestinal mucosa of naturally infected bovine hosts with clinical symptoms of JD. The protein profiles from mucosa- derived MAP were compared to the isolates grown in vitro (first culture passage). Membrane and cytoplasmic fractions were investigated separately in order to obtain a more comprehensive proteomic profile. In order to semiquantify differential protein expression between mucosa- and cultures-derived MAP, two dimensional difference gel electrophoresis (2D DIGE) was applied.
Proteome profiling of cellular fractions of host-derived MAP will provide i) further insights into MAP metabolism and pathogenesis and ii) novel targets for future work in diagnostics and vaccination.
Chapter 1 - General introduction
16 Reference List
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Mikkelsen, H., Aagaard, C., Nielsen, S. S. & Jungersen, G. (2011a). Novel antigens for detection of cell mediated immune responses to Mycobacterium avium subsp. paratuberculosis infection in cattle. Vet Immunol Immunopathol 143, 46-54.
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Nesbitt, N. M., Yang, X., Fontan, P., Kolesnikova, I., Smith, I., Sampson, N. S. & Dubnau, E. (2010). A thiolase of Mycobacterium tuberculosis is required for virulence and production of androstenedione and androstadienedione from cholesterol. Infect Immun 78, 275-282.
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Chapter 2
Material and methods
2. Chapter: Material and methods
2.1. Origin of animals and culture of M. avium subsp. paratuberculosis
Three cows of different age (2.9, 3.6 and 7.9 years) with clinical signs of Johne’s disease originating from two different farms around Giessen, Germany, were investigated (Fig. 1-1).
Fig. 1-1. Holstein–cows with clinical signs of Johne’s disease and mucosal scraping from M. avium subsp.
paratuberculosis containing intestinal peaces.
A) 2.9 years old Red Holstein cow (cow 1), B) 3.6 years old Black Holstein cow (cow 2), D) 7.9 years old Red Holstein cow (cow 3)
C) Scraping of mucosa was performed essentially as described by Choy et al. (2). Briefly, sections of frozen gut were slowly thawed overnight at 4 °C, and washed in flushing water. The mucosal gut was scraped with a microscope slide on ice.
Pieces of ileum and jejunum were obtained and cultured for the presence of MAP, and intestines were stored at –80 °C until cultural confirmation of infection.
For culture approximately 300 mg of mucosa were homogenized with glass beads (3 mm in diameter, 6 beads per tube) in 3 volumes of 0.89% saline in a Fast Prep® instrument for 30 s at intensity setting 4.0; 3 ml of the homogenized material were mixed with 7.5 ml 0.75%
A B
C D
Chapter 2 – Material and methods
26
hexadecylpyridinium chloride (HPC; Merck, Darmstadt, Germany) by vortexing and allowed to settle, 5 ml of the supernatant were added to 12.5 ml of 0.75% HPC and incubated at room temperature for 12 h. Decontaminated samples were centrifuged at 3,000 g for 15 min, and the sediment resuspended in 150 µl of residual fluid was used for cultural inoculation. A cultivation scheme is shown in Fig. 1-2.
Fig. 1-2. Cultivation of M. avium subsp. paratuberculosis.
A) Schematic workflow of cultivation, 1: Herrold’s egg yolk agar slants (HEYM); 2, 3: Oleic Acid Albumin Dextrose Complex (OADC) and Mycobactin J (Myc) supplemented Middlebrook (MB) 7H10 agar; 4: penicillin flask with 500 ml MB 7H9 broth. B) Part of a surface layer culture of MAP in a penicillin flask.
Briefly, BBL Herrold’s egg yolk agar slants containing 2 μg of mycobactin J ml−1 (Becton Dickinson, Sparks, Md.) and were incubated at 37 °C for 20 days. Subcultures were performed on Middlebrook (MB) agar 7H10 (BD Difco GmbH, Heidelberg, Germany) supplemented with 0.5% glycerol [v/v], 2 μg mycobactin J ml−1 and 10% oleic acid dextrose catalase (OADC) enrichment (1 l OADC enrichment contains 8.5 g NaCl, 50 g bovine serum albumin [BSA, fraction V], 20 g dextrose, 0.03 g catalase, 0.6 ml oleic acid) and were incubated at 37 °C for at least 40 days. For comparison with mucosa-derived MAP, large scale culture of the MAP strains isolated from mucosa was grown on 500 ml MB 7H9 broth (BD Difco) supplemented with 2.5% glycerol [v/v], 2 μg ml−1 mycobactin J and 10% OADC as surface layer in penicillin flasks at 37 °C for 70 days.
2.2. Strain typing of bovine isolates of M. avium subsp. paratuberculosis
High resolution strain typing of mucosa-derived MAP cultures was done by restriction length polymorphism (RFLP) analysis using two restriction enzymes (BstEII and PstI), multiple- locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-
> 20
HEYM 1
MB-Myc
2
> 42 d
4
8 – 12 weeks growth MB-Myc
MB-Myc
3
> 42 d
1 cm
VNTR) analysis and multilocus short sequence repeat (MLSSR) sequencing as described previously (Möbius et al., 2009).
2.3. Isolation of M. avium subsp. paratuberculosis from surface-layer cultures or directly from mucosa
MAP surface-layer cultures were grown to confluency and harvested by filtration (250 ml filtration flasks [Millipore, Schwalbach, Germany]). Bacteria were collected with a spatula and washed three times in phosphate-buffered saline (PBS; 150 mM NaCl, 9 mM Na2HPO4, 1.5 mM KH2PO4, 2.5 mM KCl, [pH 7.4]). Isolation of MAP from mucosa was performed as described by (Choy et al., 1998) with some modifications as shown in Fig. 1-3 and Fig. 1-4.
Chapter 2 – Material and methods
28
Fig. 1-3. Schematic workflow for the isolation and enrichment of M. avium subsp. paratuberculosis from gut samples.
Fig. 1-4. Pictures of M. avium subsp. paratuberculosis enrichment from intestinal mucosa.
MAP/tissue-mixture in 0.2 M sucrose layered on 0.3 M sucrose before centrifugation (1); after centrifugation (2); in 0.2 M sucrose layered on 1.5 M sucrose before centrifugation (3); after centrifugation (4); MAP pellets washed with PBS after centrifugation (5).
Briefly, sections of frozen gut were thawed overnight at 4 °C, turned inside-out and washed under flushing water. The mucosal layer was removed on ice using a glass slide, and 5 g of mucosal scrapings were homogenized in 0.2 M sucrose containing 0.1% sarkosyl and 1 mM PMSF (solution 1) for 1 min using an Ultra-Turrax. The homogenate was centrifuged (200 g, 20 min, 4 °C) and supernatants were collected on ice. The pellets were resuspended in 20 ml of solution 1 and recentrifuged (200 g, 20 min, 4 °C). All supernatants were combined and centrifuged at 8,000 g. Pellets were resuspended in solution 1, purified using a step gradient of 0.3 M sucrose (6,000 g, 10 min, 4 °C) and twice using a step gradient of 1.5 M KCl (6,000 g, 10 min, 4 °C); MAP pellets obtained were washed three times in PBS, and purity of the preparation was assessed by Ziehl-Neelsen staining and light microscopy.
2.4. Cell disruption, protein preparation of membrane-enriched and cytoplasmic fractions from M. avium subsp. paratuberculosis
Membrane-enriched and cytoplasmic fractions were prepared from mucosa- and culture- derived MAP obtained from the three cows. Three independent repeats of this enrichment were performed using bacteria from independent mucosal scrapings and cultures, respectively. Briefly, 1.2 g wet weight of the MAP pellets were resuspended in 25% sucrose, 50 mM Tris-HCl (pH 8.0) containing protease inhibitor cocktail CompleteTM (Promega, Mannheim, Germany) and mechanically disrupted with 0.1 mm zirconium beads (Biospec Products) in a Fastprep 120 (Thermo Savant) by six cycles for 20 s at 6.5 beats per second with intermittant cooling on ice. Bacterial lysates containing zirconium beads were transferred
1 2 3 4 5
Chapter 2 – Material and methods
30
to a new tube diluted with three volumes of distilled water and ultrasound-treated (three times for 1 min at setting 4, 40% duty cycle). The cell debris was removed by centrifugation (30,000 g, 10 min. at 4 °C). Supernatants were centrifuged at 100,000 g, and the pellets (MDM and CDM) were resuspended in 50 µl of standard cell lysis buffer [SCLB; 8 M urea, 2 M thiourea, 30 mM TrisHCl, 4% CHAPS (pH 8.5)]. The concentration of solubilised membrane-enriched protein fractions was determined using the 2D Quant kit (GE Healthcare, Uppsala, Sweden).
Supernatants after high-speed centrifugation contained soluble proteins mainly composing cytoplasmic proteins (MDC and CDC). Protein concentration was determined by micro BCA (Pierce-Interchim, Montluçon, France). Cytosolic proteins were concentrated by precipitation with TCA (15% v/v) overnight at 4 °C and centrifugation at 12,000 g, 15 min, 4 °C. Pellets were washed twice with 80% acetone and resuspended in an appropriate volume of standard cell lysis buffer (SCLB; 8 M urea, 2 M thiourea, 30 mM TrisHCl, 4% CHAPS [pH 8.5]) to result in protein concentrations of approximatly 10 mg ml−1. Protein samples were adjusted to pH 8.5 by titration with sodium hydroxide, protein concentrations were determined using the 2D Quant kit (GE Healthcare, Uppsala, Sweden), and treated with the PlusOne 2D Clean-Up kit (GE Healthcare) prior to use in 2D DIGE and preparative 2D gel electrophoresis (2D GE).
For a visual assessment of the preparations, 30 µg of protein extracts were separated by using SDS-PAGE and stained with Coomassie brilliant blue.
2.5. 2D difference gel electrophoresis (2D DIGE)
Independent 2D DIGE experiments were performed for comparing mucosa-derived preparations obtained from different cows and gut sections with cow-specific culture-derived preparations. An additional 2D DIGE experiment was performed comparing two cytoplasmic fractions of distinct culture-derived MAP strains. Protein preparations were labelled with Cy3 or Cy5 (GE Healthcare). For each 2D DIGE experiment, equal amounts of protein from each culture-derived preparation and its mucosa-derived counterpart were pooled and samples (each labelled with either Cy3 or Cy5) were pooled and focussed on the same Immobiline™
DryStrip for membrane fractions (24 cm, GE Healthcare, non linear, pH 3-11) and for cytoplasmic fractions Immobiline™ DryStrip pH 4-7, 24 cm (GE Healthcare). Immobiline™
DryStrips were rehydrated for 14 to 16 h using 450 µl of rehydration buffer (7 M urea [Roth, Karlsruhe, Germany], 2 M thiourea [Sigma, Deisenhofen, Germany], and 4% w/v CHAPS
[Roth]) supplemented with 2% (v/v) of the respective IPG buffer and 1.2% (v/v) of DeStreak™ reagent (GE Healthcare). The pooled protein preparations were supplemented with 1% (v/v) of the respective IPG buffer and 2% (v/v) of DeStreak™ (membrane fraction) or 2% (v/v) of DTT (GE Healthcare) (cytoplasmic fractions). Samples were loaded into anodal sample cups and subsequently focused using an Ettan™ IPGphor™ (GE Healthcare) for 21 h in the following series of time blocks with increasing voltage: 3 h at 150 V, 3 h at 300 V, 6 h at a 1,000 V gradient, 3 h at an 8,000 V gradient, and 6 h at 8,000 V. After isoelectric focussing, the strips were stored at −70°C or directly used for second dimension as previously described (Buettner et al., 2009).
2.6. Scanning, data analysis and protein identification by MALDI-TOF-MS
Differential 2D gels were scanned on a Typhoon Trio™ Scanner (GE Healthcare) at a resolution of 100 dots / cm using filters with specific excitation and emission wavelengths for Cy3 (filter BP 30; 532 nm / 580 nm), and Cy5 (filter BP 30; 633 nm/670 nm). Protein spot abundance was analyzed by DeCyder™ version 6.0 (Differential Analysis Software, GE Healthcare) using the differential in-gel analysis module (DIA). Quantification was applied for filter-confirmed spots with slope > 1.4, area < 420, and volume < 130,000. For membrane fractions, gels from 2D DIGE experiments were stained with colloidal Coomassie G-250 and prominent protein spots were excised for analysis. For cytoplasmic fractions, in parallel to 2D DIGE experiments, preparative 2D GE of respective mucosa-derived cytoplasm were performed using 0.5 mg - 1 mg of protein load. Isolated protein spots were trypsinized (Shevchenko et al., 1996), and dried samples were resolubilized in 3 µl 50% ACN, 0.1%
TFA, 1 μl of the peptide solution was mixed with 1 μl of 2 mg/ml CHCA, 50% ACN, 0.1%
TFA and spotted on the target plate. Samples were analyzed on a VoyagerDE™ Pro as described earlier (Buettner et al., 2009) or an AB Sciex ToF/ToF 5800™ mass spectrometer (both AB Sciex., Foster City, CA). 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. Mass spectrometrical data were searched against the SwissProt Database with carbamidomethylation of cysteins, oxidation of methionine and N-terminal acetylation as variable modification. A precursor mass deviation of 120 ppm and 0.5 Da for MS/MS fragments was used.
Chapter 2 – Material and methods
32 2.7. Tube-gel trypsin digestion
For tube-gel trypsin digestion (Lu & Zhu, 2005) 10 µl containing 100 µg of protein were mixed with 30 µl acrylamide-bisacrylamide solution (37.5:1; 30% [w/v]) and 30 µl distilled water containing 1% TEMED and 1% ammonium peroxodisulfate. Subsequently, proteins were fixed with 40% methanol / 10% acetic acid, and the gel was cut in small pieces. Proteins were trypsinized using Trypsin Gold-Mass Spec Grade (Promega) supplementing with 0.025% of trypsin enhancer ProteasMaxTM Surfactant (Promega) in relation of 2 µg trypsin/100 µg protein. Extraction of peptides from the gel matrix was performed in 20%, 50%, and 80% acetonitrile-water with 0.5% formic acid for 30 min in an ultrasonic bath.
2.8. SDS-PAGE-based trypsin digestion
Four of the MDC and CDC preparations (two from each isolate) were additionally analysed by gel-based liquid chromatography-tandem mass spectrometry (GeLC-MS/MS, Fig. 1-5) and tube-gel trypsin digestion followed by LC-MS/MS (Weigoldt et al., 2011). For GeLC- MS/MS, equal amounts of protein extracts (30 µg) of MDC and CDC were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie brilliant blue. From each lane seven gel slices were excised and in-gel trypsin digestion were performed as described for tube-gel trypsin digestion (see above).
Fig. 1-5. SDS-PAGE and GeLC-MS/MS of mucosa-and culture-derived cytoplasm.
10.8% (A) and 15% (B) polyacrylamide gel concentration was used for protein separation according to size.
Lanes were loaded with 30 µg protein of MDC from cow 2 (1) and cow 3 (2) as well as CDC from cow 2 (3) and cow 3 (4). Schematic workflow of GELC-MS/MS (C).
Trypsin digestion
LC - MS/MS
Search against Mycobacteria
database
97
45 30
20.1 66
kDa 1 kDa
2 3 4 5
1 2 3 4
97 45 30
20.1 66
kDa
14.5
97 45 30
20.1 66
14.5
6 7
kDa
1 2 3 4A
B
C
97
45 30
20.1 66
Chapter 2 – Material and methods
34
2.9. Nanoflow-liquid-chromatography-coupled tandem mass spectrometry (nUPLC- ESI Q-TOF-MS/MS)
A 100 µm x 100 mm nUPLC column (ACQUITY UPLC® 1.7 µm BEH130 C18; Waters, Milford, MA, USA) was used for peptide separation. Each sample was run over 45 min with a flow rate of 300 nl min-1. Mobile phase A consisted of 0.1% formic acid in 1% acetonitrile.
Mobile phase B included 0.1% formic acid in 99% acetonitrile. The gradient was 99% mobile phase A for 0.33 min and then ramped linearly to 65% of mobile phase A over 30 min. Over the next 1 min, it was ramped to 15% phase A and held for 1 min before equilibrating the column with 99% phase A for 13 min. Before each other run, calibration of the Quadrupole Time-Of-Flight tandem mass spectrometer (Q-TOF Ultima, Waters) was performed using [Glu1]fibrinopeptide B as standard. The capillary voltage was 1800 V and was tuned for signal intensity. The two most intense ions with charge states between 2 and 4 and mass ranges between 450 and 1600 were selected in each survey scan if they met the switching criteria. Different collision energies were used to fragment each peptide ion on the basis of its mass-to-charge (m/z) values. For tube-gel trypsin digestion, each sample was run up to eight times. For each new measurement, previously detected peptides in a mass range within ±100 mDa were excluded from analysis in order to identify fragmentation on peptides represented in weaker signals. By this approach each sample was measured until no further increase in the number of identified peptides was observed. The search algorithm was set to allow for carbamidomethylation on cysteine residues, oxidation on methionine residues, and a maximum of one missed cleavage. A minimum of one validated peptide containing four or more consecutive y-ions was set for protein matches.
2.10. High throughput protein analysis using nanoflow-liquid-chromatography-coupled tandem mass spectrometry (LC-orbitrap-MS/MS)
The LC-orbitrap-MS has been applied in corporation with Prof. Dr. rer. nat. Andreas Pich, MS lab in the Institute for Toxicology, Medical School Hannover.
Peptide extracts were combined, dried and redissolved in 10 μl 2% ACN, 0.1% TFA. LC- MS/MS analysis was performed on an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) as described recently (Boer et al., 2011). Briefly, an appropriate sample amount was loaded onto a nanoflow ultra-high pressure liquid chromatography system (RSLC, Dionex) equipped with a trapping column (5 mm C18 particle, 75 mm ID, C18
material, 2 cm length, PepMap, Dionex) and separating column (2 mm C18 particle, 75 mm ID, 50 cm length, PepMap, Dionex). After trapping, peptides were eluted with a linear gradient of buffer B (80% ACN, 0.1% formic acid) in buffer A (0.1% formic acid) from 4%
to 25% in 60 min, 25%-50% in 25 min and 50%-90% in 5 min, after which the column was flushed for 10 min isocratically with 90% B and reconditioned to 4% B in 20 min. Flow rate was 250 nl/min with a column temperature of 40 °C. Peptides were ionized in the nanoESI source with 1.2 kV. Overview scans were acquired at a resolution of 60 k in a mass range of m/z 300-1600 in the orbitrap. The top 10 most intensive ions of charge two or three and a minimum intensity of 2000 were selected for CID fragmentation with a normalized collision energy of 38.0, an activation time of 10ms and an activation Q of 0.250 in the linear ion trap mass analyzer of the LTQ Orbitrap Velos. Fragmentation mass spectra were also recorded in the LTQ. The m/z values in a 10 ppm mass window of the selected ions were subsequently excluded from the fragmentation for 70 s. Data analysis was facilitated by proteome discoverer software 1.2 (Thermo Fisher Scientific, Langenselbold, Germany) and the Mascot search algorithm. Mascot was set up to search a customized database generated using the UniProt database (release 2012_03). It includes M. avium subspecies paratuberculosis 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 identified if at least two unique peptides were detected.
2.11. Criteria for protein identification and differential expression
Proteins from mucosa- and culture-derived MAP were considered as identified if they were detected at least twice by GeLC-MS or tube-gel trypsin digestion followed by MS in independent biological repeats. Alternatively, proteins were also considered as identified if isolated from 2D gels and giving a significant database match of the peptide mass fingerprint.
The proteins identified in MDCs were considered to be present also in CDCs after a single detection by GeLC-MS or tube-gel trypsin digestion followed by MS.
Proteins were considered as differentially expressed only if an at least 1.5-fold increase in expression could be observed in MDC preparations from each of the two cows in comparison to the corresponding CDCs.
Chapter 2 – Material and methods
36 2.12. Data processing and bioinformatics
LC-MS/MS raw data were processed using ProteinLynx™ Global Server (Version 2.1, Waters) by searching against the Mycobacterium species SWISS-PROT database downloaded on 25.09.2008 [99,308 entries]. The identification of bovine proteins was performed by searching against the NCBI nonredundant database as downloaded on August 16th 2006. All proteins identified in MDM or CDM as being specific for MAP were copied into separate Microsoft Excel data sheets. The mycobacterial origin of all proteins detected in MDM by a one peptide-hit only was confirmed by searching against the entire NCBI data base.
Prediction for membrane association or cytoplasmatic localisation of these proteins was performed with PSORT (http://psort.ims.u-tokyo.ac.jp/form.html). Verified datasets were organized according to their distribution in the Cluster of Orthologous Groups (COGs).
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 KEGG database was searched using MAP annotation numbers and M. tuberculosis orthologues 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) and NCBI.
Reference List
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3. Chapter:
Differential proteome analysis of Mycobacterium avium subsp.
paratuberculosis grown in vitro and isolated from cases of clinical Johne’s disease
Mathias Weigoldt1, Jochen Meens1, Klaus Doll2, Isabel Fritsch3, Petra Möbius3, Ralph Goethe1†, Gerald-F. Gerlach1†‡
1 Institute for Microbiology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Hannover, Germany
2 Clinic for Ruminants and Swine (Internal Medicine and Surgery), Justus-Liebig-University, Giessen, Germany
3 Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Jena, Germany
† These authors contributed equally to this work.
‡ Corresponding author. Current address: IVD GmbH, Heisterbergallee 12, 30453 Hannover, Germany.
Correspondence Gerald-F. Gerlach gfgerlach@gmx.de
Running title: Johne’s disease - bacterial proteins in the host Contents category: Microbial Pathogenicity
Keywords: M. avium subsp. paratuberculosis; differential protein expression; protein expression in the host; 2D DIGE; nUPLC-ESI Q-TOF-MS/MS
(Manuscript has been published in Microbiology 2011, 157, 557-565) (Online available: doi: 10.1099/mic.0.044859-0)
