2. Materials and Methods

2.1. Bacterial strains and culture methods


E. coli was transformed by conventional heat shock transformation or electroporation and plated on LB-agar (Invitrogen, Paisley, UK) containing appropriate antibiotics (hygromycin B [Roche, Mannheim, Germany] at 150 µg/ml, kanamycin [Sigma Aldrich, St. Louis, MO, USA] at 35 µg / ml or ampicillin [ICN, Aurora, OH, USA] at 100 µg / ml). For blue-white selection, 1 mM IPTG (Gerbu, Gaiberg, Germany) and 75 µg / ml X-Gal (Roth, Karlsruhe, Germany) were added. All cloning procedures were done in E. coli DH5α (Invitrogen) unless stated differently. Liquid E. coli cultures were grown in LB-medium (Invitrogen) containing appropriate antibiotics (hygromycin B at 150 µg/ml, kanamycin at 50 µg/ml or ampicillin at 100 µg/ml). M. smegmatis mc2155 was electroporated as described [40] and plated on Middlebrook 7H10 agar (BD, Franklin Lakes, NJ, USA) supplemented with 10 % albumin-dextrose saline (ADS: 0.81 % NaCl, 5 % BSA Fraction V [Serva, Heidelberg, Germany], 2 % glucose), 0.5 % glycerol, 0.05 % Tween-80 (Sigma) and either hygromycin B (50 µg/ml) or kanamycin (25 µg/ml). Liquid M. smegmatis cultures were grown in Middlebrook 7H9 medium (BD) supplemented with 10 % ADS, 0.05 % Tween-80 (Sigma) and 0.2 % glycerol (further referred to as 7H9 complete medium), containing either hygromycin B (50 µg/ml) or kanamycin (25 µg/ml). If required, sucrose was used at a concentration of 2 % and added after the medium had been autoclaved and cooled to 55°C. Biofilms were generated in M63 salts minimal medium. One liter M63 medium consisted of 13.6 g KH2PO4, 3.8 g (NH4)2SO4, 0.5 mg FeSO4 . 7 H2O, 0.2 % carbohydrate, 1 mM MgSO4, 0.7 mM CaCl2, 0.5 % casamino acids (BD) adjusted to pH 7.0 (further referred to as biofilm medium).


[page 21↓]

2.2.  Construction of the M. smegmatis knockout template


The plasmids used in this study are listed in Table 5. Plasmids were amplified in E. coli HB101 or E. coliDH5α and purified with Qiagen columns as recommended by the manufacturer (Qiagen, Inc., Chatsworth, CA, USA). DNA fragments used for plasmid construction were purified by agarose gel electrophoresis and recovered by QIAEXII gel extraction kit (Qiagen).
Mycobacterial genomic DNA was prepared from 10 ml cultures. Bacteria were lysed with 1.3 ml of a 3:1 mixture of chloroform-methanol. The lysate was mixed with 1.3 ml of Tris-equilibrated phenol and 2 ml of RLT buffer (Qiagen) supplemented with 0.5% N-lauroylsarcosine (Sigma) and 1% β-mercaptoethanol. The upper phase was collected after centrifugation, and the genomic DNA was precipitated with isopropanol. This DNA preparation method was also scaled down to the use of 1 / 10 of all ingredients and thus was called microprep. For southern blotting, genomic DNA was digested with BamHI and transferred to a positively charged nylon membrane (Roche) using a pressure blotter. The probe was labeled and fragments were visualized with DIG DNA Labeling and Detection Kit (Roche) as recommended by the manufacturer.
The in-frame, unmarked deletion template Δupk contains the 2 flanking regions next to upk. Flanking regions (879 bp and 945 bp) and the M. tuberculosis upk homologue rv2136c were amplified by PCR. Specific primer pairs and chromosomal DNA are listed in Table 5. PCR was carried out for 1 initial cycle of 5 min at 95°C, followed by 25-30 cycles (95 °C for 1 min, 60°C for 1 min, 72 °C for 3 min), and 1 final cycle at 72 °C for 10 min. All PCR products were blunt end cloned into the SrfI site of pPCR-Script SK+ Amp. The respective cloning kit was used (Stratagene, La Jolla, CA, USA). Sequence identity was verified by [page 22↓]sequencing. The flanking regions were cleaved from cloning vectors using HindIII and EcoRV. In a 2 step ligation, flanking regions 1 and 2 were first dimerized and then inserted into the EcoRV predigested and dephosphorylated vector pYUB657.

2.3. Electron microscopy (performed in collaboration by Dr. Volker Brinkmann)


For fine structural analysis, cells were fixed with 2.5 % glutaraldehyde, postfixed with 1 % osmiumtetroxid, contrasted with uranylacetate and tannicacid, dehydrated and embedded in Polybed (Polysciences, Eppelheim, Germany). After polymerization, specimens were cut at 60 nm and contrasted with lead citrate. For immunodetection, cells were fixed with 4 % PFA and embedded in a mixture of 25 % sucrose / 10 % polyvinyl alcohol (PVA). Ultrathin sections were cut at –105 °C, blocked, reacted with primary anti-peptidoglycan-antibodies (Biotrend, Köln, Germany) followed by secondary antibodies coupled to 6 nm gold particles. Specimens were analyzed in a Leo 906E transmission electron microscope.

2.4. Alamar blue assay


Antimicrobial susceptibility testing of bacitracin (Calbiochem, San Diego, CA, USA) and Isoniazid (Sigma) was performed in 96-well microplates. Outer perimeter wells were filled with sterile water to prevent dehydration in experimental wells. Antibiotic dilutions were dissolved in distilled deionized water and subsequent dilutions were performed in 7H9 complete medium. Wildtype, mutant, and the reconstituted strain of M. smegmatis mc2 155 or M. tuberculosis H37Rv each were inoculated in 7H9 complete medium and grown at 37°C [page 23↓]until OD600 = 1.0 was reached. This culture was diluted 1 : 40 and 100 µl per well were added to a microtiter plate containing titrated antibiotics. Plates were covered with breathable sealing membrane (Nunc, Naperville, IL, USA) and incubated at 37 °C for 16-20 h in the case of M. smegmatis and 4 to 5 days in the case of M. tuberculosis. Then 5 µl per well of Alamar Blue (Serotec, Oxford, UK) was added. After further 6-7 h (M. smegmatis) incubation at 37°C, extinction at 570 nm was measured in an ELISA-reader. Respectively, after over night (M. tuberculosis) incubation pictures were taken to document. Since there was no elisa-reader in the S3 facility, color change was examined visually to determine value of bacterial growth.

2.5. Biofilm formation


Single colonies of M. smegmatis mc2 155 wildtype and M. smegmatis Δupk mutant were inoculated in 7H9 complete medium and grown to saturation. Fifty µl from this preculture were inoculated into 1 ml of biofilm medium and cultured in 12 well plates (Nunc) at room temperature (RT) for 4 to 5 days. The medium was removed and 500 µl of 1 % crystal violet (BD) were added. Plates were incubated at RT for 30 min, rinsed with water and examined under the microscope. For microscopy, biofilms were grown on glass cover slips, which had been placed into each cavity of a 12 well plate. Auramine Rhodamine staining was performed with Fluorescent Stain Kit B according to the recommendations of the manufacturer (BD).
Male C57BL/6 mice were used for the in vivo biofilm assay. Ten mice per group were anesthetized with 50 µl of a 1:1:3 mixture of Ketavet (Pharmacia, Erlangen, Germany), Rompun (Bayer, Leverkusen, Germany), and PBS. Subsequently 1 x 107 bacteria / 5 µl were applied to a penis. Next day, overnight grown smegmata were counted. Mice wore protections manufactured from 50 ml tubes (BD), to avoid cleaning.


[page 24↓]

2.6.  Infection of bone marrow derived mouse macrophages


Bone marrow cells from C57BL/6 mice were flushed out of femura and tibiae using a syringe with a 23G gauge (Braun, Melsungen, Germany). Differentiation and harvesting was performed as described before [41]. Infection was executed in 24 well plates using RPMI medium (Biochrom, Berlin, Germany) supplemented with 10 % FCS (Biochrom), 1 % Hepes (Biochrom), and 1 % L-Glutamine (Biochrom). One million bone-marrow derived macrophages were used per well. M. smegmatis was added at a multiplicity of infection (MOI) of 200. Infection lasted for 2 h, followed by 3 washes with RPMI supplemented with 10 % FCS, Hepes, L-Glutamine, and 250 µg Amikacin / ml (Sigma). Subsequently, cells were lysed with 1 ml 0.1 % TritonX100 at different time points and serial dilutions were plated on 7H10 agar plates.

2.7. Construction of a recombinant TM4 knockout phage


Flanking regions to the target gene upk were cloned into the vector pJSC284 next to a hygromycin resistance cassette. The sizes of the flanking regions were: flanking region 1 = 810 bp and flanking region 2 = 807 bp amplified by PCR (conditions: see above; Primers: Table 5) and extended with the restriction sites BamHI (flanking region 1) and AgeI (flanking region 2). The PCR products were purified from an agarose gel and ligated in pPCR-Script Amp SK(+) (Stratagene), transformed, amplified, and sequences were verified. Flanking region 2 had an internal AgeI restriction site which was overlooked in the beginning. To overcome this problem, the fragment was No tI-BamHI-cut out of pPCR-Script and blunted by Klenow reaction, performed as the manufacturer recommended (NEB). Subsequently fragments were ligated into the BamHI blunted AgeI restriction site of pJSC284. The resulting vector was called pKO-upk (Fig. 4). For amplification it had to be transformed into E. coli [page 25↓]HB101 as the DH5α strain exhibits resolvase activity which may affect plasmid stability. The pKO-upk and the genome of TM4 phage phAE159 were PacI digested in order to fuse the knockout construct to the phage genome. Before, genomic phage DNA had to be self-ligated via internal cos-sites. Because of a very inefficient phAE159-Δupk producing ligation reaction, DNA was in vitro packaged into λ-phage-heads using Gigapack III (Stratagene). Transduction-competent E. coli HB101 were used, and phasmid containing bacteria were selected on hygromycin containing LB-agar plates. As phasmids are huge molecules, DNA was purified by classical alkaline lysis miniprep. Control for correct insertion of the knockout construct, was performed by PacI digest, which revealed a 5 kb fragment by gel electrophoresis. All positive clones were pooled. One µl of recombinant phasmid DNA was used to transform M. smegmatis as a host for phage production. Phages had to maintain temperature sensitivity (ts) so that they became lytic at 30°C but not at 37°C. This was critical because subsequently transduced M. tuberculosis was to be grown at 37°C and should not have succumbed to lysis.

Electroporation conditions for transformation of mycobacteria, in this case M. smegmatis, were:

1000 Ω
25 µF
2.5 kV

A cuvette with a 0.2 cm gap was used. This was followed by addition of 1 ml 7H9 complete medium, 30 min incubation at 30°C and plating in 2 alternative ways:

[page 26↓]1. Electroporation of 900 µl M. smegmatis solution, mixed with 4 ml top-agar and poured on mycobacteriophage plates. This was working well in the case of inefficient transformation.

2. Electroporation of 100 µl M. smegmatis solution, mixed with 100 µl M. smegmatis wildtype solution and 4 ml top-agar poured on mycobacteriophage plates. This worked well in the case of a very efficient transformation that was in need of more bacteria for lysis.

mycobacteriophage plates:

19 g 7H10 agar (BD)
1000 ml H2O
autoclaved, and further enriched with 10 ml 20 % dextrose and 10 ml 50 % glycerin

top agar:

0.235 g 7H9 medium powder (BD)
0.38 g noble agar (BD)
50 ml H2O
autoclaved, liquefied in a microwave oven before use and enriched with 500 µl 20 % dextrose. Used in 4 ml aliquots that were kept liquid at 50°C in a heat block.

Plates were incubated 3 to 4 days at 30°C. During this time a lawn of M. smegmatis was growing in the top-agar, intermitted by plaques, produced by lytic phages. Plaques were picked and patched on 2 top-agars which were subsequently incubated at 30°C or 37°C to [page 27↓]assess whether the phages had retained their temperature-sensitivity. One ts phage was picked and eluted over night at 4°C in 500 µl of mycobacteriophage-buffer (MP-buffer).

MP-buffer:

25 ml 1 M Tris / HCl, pH 7.5
75 ml 1 M NaCl
5 ml 1 M MgSO4
1 ml 1 M CaCl2
H2O ad 500 ml
autoclaved, or filter sterilized

This extract was called “phage-lysate”. To amplify the phage, 300 µl of serial phage-lysate dilutions (undiluted to 1 x 10-4) in MP-buffer were mixed with 300 µl of M. smegmatis and adsorbed at 30°C for 30 to 120 min. Out of each dilution-mix, 3 times 200 µl were mixed with 4 ml top-agar, poured on mycobacteriophage plates, and incubated 3 to 4 days. To harvest the phages, plates were chosen in which the phage had lysed almost the entire lawn (“lacy plates”). Top agars were collected with cell scrapers (Sarstedt, Newton, USA) and incubated over night with 2 - 4 ml MP-buffer per top agar at 4°C. This was followed by 10 min centrifugation at 4000 rpm, and 0.22 µm filter sterilization of supernatants. To determine the titer of the phage lysate, dilutions up to 10-9 were prepared in MP-buffer, and 5 µl per dilution were spotted on a mycobacteriophage plate with a top-agar containing 200 to 400 µl M. smegmatis. After 3 to 4 days, plaques per spot were counted, and the titers calculated. A last verification of proper orientation of the flanking regions and possession of the [page 28↓]hygromycin resistance cassette was done by PCR using flanking region- and HygOut- primers and 1 x 10-2 dilutions of the phage lysate.

Fig. 4 pKO-upk, the vector to be fused to the TM4 phage genome of phAE159. Flanking regions 1 and 2 were cloned next to the hygromycin resistance cassette.

2.8. Transduction of M. tuberculosis


A culture of M. tuberculosis H37Rv was grown to OD600 = 0.8 – 1.0. Per transduction, 10 ml culture were centrifuged at 3000 rpm for 10 min and the pellet was resuspended 1 ml MP-buffer. One ml of a high titer phage lysate (at least 1010 pfu/ml) was pre-warmed at 37°C, mixed with the concentrated bacteria, and incubated for 4 h at 37°C. Subsequently, transduced cells were centrifuged for 10 min at 3000 rpm, resuspended in 500-1000 µl 7H9 medium, and plated on 4 7H10 plates containing 75 µg hygromycin / ml. After 4 weeks of incubation at 37°C, clones were picked to inoculate 5 ml liquid cultures in 7H9 complete medium shaking at 90 rpm for 1-2 weeks. Following, medium was added to expand the cultures. Ten ml were used to prepare chromosomal DNA for southern blot analysis of the clones.


[page 29↓]

2.9.  Reconstitution of the mutants


The upk gene was amplified by PCR (Primers: see Table 5), cloned into pPCRScript, sequenced, and cloned into the E. coli mycobacteria shuttle vector pMV262 under control of the M. bovis groEL2 (hsp60) promoter as translational fusion. Competent M. tuberculosis H37Rv Δupk were electroporated, and plated on 7H10 agar plates containing 75 µg hygromycin per ml and 25 µg kanamycin per ml. After 4 weeks, clones were picked, and 5 ml cultures were inoculated. Out of well-grown cultures, 1 ml was taken to perform DNA microprep and subsequent PCR to check for the kanamycin resistance cassette and the upk gene. DNA derived from a positive clone was also taken to transform E. coli DH5α. The amplified and purified plasmid was

Fig. 5 Reconstitution vector pMV262-upk. The upk gene was cloned into the BamHI restriction site under control of the groEL2 promoter.


BamHI digested and compared by agarose gel electrophoresis to the original plasmid. Transcription of the upk genefrom the reconstitution vector pMV262-upk was quantified by real-time PCR which is based on the measurement of amplified products after each cycle of [page 30↓]the PCR using fluorescent dyes interacting only with double stranded DNA. The more template that is present at the beginning of the reaction, the lower the number of cycles it takes to reach a point in which the fluorescent signal is first recorded as statistically significant above background, which is the definition of the threshold cycle (Ct). Comparison of the threshold cycle for a specific template in each sample leads to semi-quantitative evaluation of original template concentration. For semi-quantitative real-time PCR total RNA was transcribed to cDNA using SuperscriptTM III (Invitrogen) as the manufacturer recommended. All PCRs were run for 40 cylces with 20 sec 94°C and 60 sec 60°C in the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using ABI PRISM optical 96-well plates (Applied Biosystems). Primers are listed in Table 5. Reaction mixtures were set up in 30 µl final volume using 15 pmol of each primer, 5 µl template cDNA and 15 µl 2x SYBR-Green PCR Master mix (Applied Biosystems). Data analysis was performed using the ABI Prism 7000 SDS Software and MicrosoftExcel.

2.10. Preparation of competent E. coli


Five ml of Psi medium was inoculated with one colony and precultured over night at 37°C.

Psi medium:

2.5 g Bacto Yeast Extract (BD)
10 g Bacto Tryptone (BD)
2.5 g MgSO4
[page 31↓]500 ml H2O
adjusted with KOH to pH 7.6 and filter sterilized

One hundred ml of Psi medium were inoculated with 1 ml of the preculture and grown at 37°C to OD550 = 0.5. Thereupon the culture was left 15 min on ice, centrifuged for 10 min at 3000 rpm and resuspended in 40 ml cold TfbI solution.

TfbI solution:

30 mM potassium acetate
100 mM rubidium chloride
10 mM calcium chloride
50 mM manganese chloride
15 % v/v glycerol
H2O ad 200 ml
adjusted to pH 5.8 with diluted acetic acid and filter sterilized

After 15 min on ice, and centrifugation for 10 min at 3000 rpm, the pellet was resuspended in 4ml of cold TfbII solution.

TfbII solution:

10 mM MOPS
75 mM calcium chloride
10 mM rubidium chloride
15 % v/v glycerol
[page 32↓]H2O ad 100 ml
adjusted to pH 6.5 with diluted caustic soda and filter sterilized

After further 15 min on ice, cells were used immediately or were frozen in liquid nitrogen and stored at –80°C. For each transformation 40 - 50 µl of the cells were mixed with up to 1 µg DNA, incubated for 30 min on ice, 45 s at 42°C, and again 2 min on ice. One ml of SOC medium was added and after 1 h at 37°C, cells were plated.

2.11. Purification of chromosomal DNA from mycobacteriophages


Five µl of DNAse (1 mg / ml) and 10 µl RNAse (10 mg / ml) were added to 1 ml of phage-lysate, incubated for 30 min at 37°C, and supplemented with 50 µl of freshly made STEP-buffer.

STEP-buffer:

400 mM EDTA pH 8.0
50 mM Tris pH 8.0
1 % SDS
H2O
EDTA was pre-warmed to 50°C, and complete STEP-buffer was kept at 50°C to avoid SDS precipitation.
After addition of 20 µl of proteinase K (10 mg / ml) (Merck, Darmstadt, Germany) and 30 min incubation at 56°C, the sample was split. Each part was 2 times extracted with 500 µl phenol and 2 times with 500 µl chloroform : iso-amyl-alcohol (24:1). Supernatants were [page 33↓]mixed with 50 µl of sodium acetate and 1 ml of 100 % ethanol. After 2 min at RT, DNA was precipitated by spinning for 20 min at 13,000 rpm. The pellet was washed with 1 ml 70% ethanol, air-dried and resuspended in 50 – 100 µl TE-buffer over night at 4°C. A few µl on an agarose gel demonstrated a band at 50 kb.

2.12.  In vitro packaging of phasmid DNA


Two µl of a phasmid ligation were mixed with an aliquot of Gigapack III Gold (Stratagene), briefly centrifuged and incubated at RT for 90-120 min. Afterwards 500 µl of SM-buffer were added. This solution was called packaging mix.

SM-buffer:

0.58 g NaCl
0.2 g MgSO4 x 7 H2O
5 ml Tris / HCl, pH 7.5
10 mg gelatine or 0.5 ml of a 2 % solution
H2O ad 100 ml
autoclaved

Fifty µl of the packaging mix were incubated with 200 µl of transduction competent E. coli HB101 for 20 min at 37°C without shaking. After addition of 1 ml LB-medium and 60 min expression at 37°C, bacteria were plated on LB-plates containing 150 µg of hygromycin and grown over night at 37°C.


[page 34↓]

2.13.  Preparation of transduction competent E. coli HB101


Twenty ml LB-medium which had been enriched with 0.4 % maltose and 10 mM MgSO4 were inoculated with 500 µl of a well grown E. coli HB101 preculture, and grown to OD600 = 0.5 – 0.7 at 37°C. Ten ml were centrifuged at 3000 rpm for 10 min, and the pellet was resuspended in 5 ml of 10 mM MgSO4.

2.14. Classical miniprep


The pellet of a 5 ml E. coli culture was resuspended in 100µl of LB-medium. Cells were lysed in 300 µl TENS-solution and incubated for up to 5 min at RT.

TENS-solution

94 ml sterile TE-buffer
1 ml 10 M NaOH
5 ml 10 % SDS

TE-Buffer

5 ml 1 M Tris/HCl, pH 8.0
1 ml 0.5 M EDTA, pH 8.0
H2O ad 500 ml
autoclaved

[page 35↓]This was mixed with 150 µl sodium acetate (pH 5.2), incubated for 5 – 10 min on ice, and centrifuged for 10 min at 13,000 rpm. The supernatant (about 500 µl) was transferred to a new reaction tube, and mixed with 500 µl phenol : chloroform : iso-amyl-alcohol (25 : 24 : 1). After 5 min centrifugation at 13,000 rpm the supernatant was transferred to a new reaction tube, 900 µl 100 % ethanol was added, followed by 1 min incubation at RT and 15 min centrifugation at 13,000 rpm. The pellet was washed with 1 ml 70 % ethanol, dried, and resuspended in 50 µl TE-buffer enriched with 1 µl RNAse (10 mg / ml).

2.15. Preparation of electro-competent mycobacteria


Fifty ml M. tuberculosis respectively M. smegmatis was grown in 7H9 complete medium to OD600 = 0.6 - 1.0. M. smegmatis was cooled for 1-2 h on ice and subsequently washed 3 times with cold 10 % glycerol. The pellet was resuspended in 2 ml cold 10 % glycerol. In case of M. tuberculosis, bacteria were washed 3 times with 10 % glycerol at RT and resuspended in 5 ml 10 % glycerol.

2.16. Neutral-red staining


Five ml of a well grown bacterial culture were centrifuged at 3000 rpm for 10 min, supernatant was discarded, 5 ml 50 % aqueous methanol added, followed by incubation for 1 h at 37°C. Again, the bacteria were washed with 50 % aqueous methanol, and the pellet was resuspended in 5 ml neutral-red (Merck).

neutral red:

1 vial barbital buffer (Sigma)
[page 36↓]2 g neutral red
ad 1000 ml
pH 9.8
sterile-filtered

Color change was visible after 1h at RT.

2.17. Pellicle formation


Standing cultures were used to investigate pellicle formation of the M. tuberculosis strains. Therefore 20 ml Sauton medium was inoculated with 1 ml of a well grown pre-culture, incubated in 50 ml conical tubes at 37°C.

Sauton medium:

20 g L-asparagine
10 g sodium-citrate
2.5 g K2HPO4
2.5 g MgSO4 x 7 H2O
250 mg ammonium-iron-III-citrate
300 g glycerin
ad 5 l H2O
sterile-filtered

2.18. Cording assay


About 3 µl of a well-grown culture were transferred into subdivisions of a 8 chamber-slide [page 37↓](Nunc), and air dried. Subsequently 1 ml of 7H9 complete medium without Tween 80 was added, followed by 2 – 3 weeks incubation at 37°C. Then, medium and chambers were removed, and the slide was soaked in formalin over night. Staining was performed with the TB Fluorescent Stain Kit B (Auramine O-Rhodamine B) (BD) as the manufacturer recommended.

2.19. RNA-preparation from mycobacteria and analysis of the gene expression pattern (performed in collaboration with Dr. Helmy Rachman)


RNA-preparation was performed in conformity with isolation of genomic DNA (see above) as this procedure allows purification of all nucleic acids. Purification of RNA from the aqueous phase was performed using RNeasy Mini Kit (Qiagen) as the manufacturer recommended. Labeling, hybridization to a DNA-array, scanning and evaluation of the signals were done in collaboration with Helmy Rachman and is described in his thesis “Functional Genome Analysis of Mycobacterium tuberculosis”, 2003.

2.20.  Preparation of M. tuberculosis whole cell lysates for two-dimensional electrophoresis (2-DE)


A culture of 100 ml M. tuberculosis was grown to OD600 = 0.8 – 1, centrifuged at 3000 g and 4°C for 15 min, washed twice with 100 ml cold PBS 0.05 % Tween 80 resuspended in 1 ml cold PBS Tween 80, transferred to a micro-centrifuge screw cab tube with lid gasket, and centrifuged at 4 °C and 10,000 g for 15 min. To avoid proteolytic degradation, 1 µl of each of the protease inhibitors TLCK (100 mg / ml), pepstatin A (50 mg / ml), leupeptin (100 mg / ml) and E64 (25 mg / ml) diluted in DMSO was added to the cell pellet, which was sonified [page 38↓]afterwards. Urea was gradually added to a final concentration of 9 M to the sonicate (108 mg urea / 100 µl sonicate). Furthermore, dithiothreitol (DTT) to a final concentration of 70 mM, ampholytes (Servalytes 2 – 4; 2 %) and Triton X-100 (2 %) were added. The sample was kept at RT for 30 min, stirred occasionally, centrifuged at 10,000 rpm and 16°C for 15 min. The supernatant was removed and 2-DE performed.

2.21. Protein separation by two-dimensional electrophoresis (performed in collaboration with Dr. Jens Mattow)


Protein separation by 2-DE was performed as a combination of carrier ampholyte isoelectric focusing (IEF) and SDS-PAGE using gels with a size of 23 x 30 cm [42]. IEF was performed in rod gels containing 9 M urea, 3.5% acrylamide, 0.3% piperazine diacrylamide and a total of 4% ampholytes pH 2-11 (Servalytes 2-11; Serva, Heidelberg, Germany). Protein samples were applied at the anodic side of the IEF gels and focused under non-equilibrium pH gradient electrophoresis conditions (8,870 Vh). For analytical and preparative investigations 0.75 mm or 1.5 mm thick gels were used, respectively. For analytical investigations, 60 μg of protein sample were applied. For preparative experiments, we applied up to 600 μg of protein sample. SDS-PAGE was performed in gels containing 15% acrylamide using the IEF gels as stacking gels. Following electrophoresis, proteins were visualized by either silver (analytical gels) [43] or Coomassie Brilliant Blue (CBB) G250 staining (preparative gels) [44]. The pI and Mr gradient of the 2-DE gels was determined using an iterative calibration method as described [45].


[page 39↓]

2.22.  Evaluation of differential proteins by PDquest (performed in collaboration with Dr. Jens Mattow)


The 2-DE patterns were first examined visually aimed at the identification of spots of differential relative intensity between whole cell lysates of wild-type M. tuberculosis H37Rv on the one hand and the deletion mutant M. tuberculosis H37RvΔupk on the other hand. For both strains, 3 different protein samples were prepared, and two 2-DE runs were performed per sample. To elucidate potential variations, two 2-DE gels of independently prepared samples of M. tuberculosis H37Rv were compared individually with 2 patterns of the mutant strain. Variants which were detected in both gel comparisons were regarded as potential variants. These were finally checked by analyzing all 2-DE patterns, i.e. 6 patterns per strain, and only stringently confirmed differences were accepted as specific variations. For quantitative analysis, the 2-DE gels were further evaluated using the image analysis software programme PDQuest (Version 7.1, BioRad, Hercules, CA, USA). After scanning the gels (8 bit gray values; 100 dpi), spot detection and quantification were performed automatically by fitting spot intensities with a two-dimensional Gaussian model. Corresponding spots in distinct 2-DE gels were matched using a distortion model that takes into account local gel running differences. Prior to the final quantitative data analysis, spot detection and matching were checked thoroughly and corrected manually in an interactive manner. This time-consuming step was essential to achieve reliable results. For quantitative data analysis of spot intensities, a t-test (significance level P<0.05) was applied.


[page 40↓]

2.23.  Protein identification by mass spectrometry (performed in collaboration with Dr. Jens Mattow)


Identification of gel-separated proteins was performed using matrix assisted laser desorption/ionisation mass spectrometry (MALDI-MS) peptide mass fingerprinting (MALDI-MS PMF) [46,47,48,49], electrospray ionisation tandem mass spectrometry (ESI-MS/MS) [49] and/or capillary liquid chromatography in combination with ESI-MS/MS (CapLC-MS/MS) [50]. MALDI-MS PMF was performed as described [51,52] with minor modifications. In short, spots of interest were excised from preparative CBB G250-stained 2-DE gels and proteins were digested in-gel using trypsin. Resulting peptides were desalted and concentrated prior to mass analysis using ZipTipC18 pipette tips (Millipore, Bedford, USA). Masses of the tryptic peptides were determined using a time-of-flight mass spectrometer with delayed extraction. Mass accuracy in the range of 30 ppm was obtained by internal calibration of the spectra. Proteins were identified by PMF using the search algorithm MS-FIT. The applied presettings and criteria for identification have been described previously [53]. In some cases, sequence support was required, to establish protein identity. Sequence support was obtained by either ESI-MS/MS performed as described [54] or CapLC-MS/MS as described below. For ESI-MS/MS analyses the sequence tag method [55] was used to search for the proteins in the NCBI protein database (http://195.41.108.38/PA_PeptidePatternForm.html). If this was not successful, a de novo sequencing with the program MassSeq (Micromass, Manchester, UK) and a database search in the M. tuberculosis protein database of the Institute for Genomic Research (http://www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database=gmt) were performed. The search was carried out for the coding part of the genome and the entire genome. Some peptide mixture samples were [page 41↓] chromatographically separated prior to on-line mass analysis using a capillary liquid chromatography system delivering a gradient to formic acid (0.1%) and acetonitrile (80%). The eluted peptides were ionized by electrospray ionization on a Q-TOF hybrid mass spectrometer. The instrument, in automated switch mode, selects precursor ions based on intensity for peptide sequencing by collision-induced fragmentation tandem MS. The MS/MS analyses were conducted using collision energy profiles that were chosen based on the m/z value of the precursor. The generated mass data were processed into peak lists containing m/z value, charge state of the parent ion, fragment ion masses and intensities, and correlated with proteins and nucleic acid sequence databases using the search algorithm Mascot [56]. Proteins were identified based on matching the MS/MS data with mass values calculated for selected ion series of a peptide. A non-redundant protein database and a nucleotide database (dbEST) were searched without applying any constraints on Mr or species. Results were validated manually .

2.24. Infection procedures


Before intranasal infection, mice were anesthetized according to animal protection law with 10 µl (0.2 mg) Rompun (Bayer), and 10 µl (1.2 mg) Ketavet (Pharmacia). Thirty µl PBS was added, and a total volume of 50 µl / mouse was injected into the femoral muscle. After a few minutes mice were anesthetized, and the appropriate dose of bacteria was given as drops of 20 µl on the nostrils.
Intravenous infections (0.1 ml) were given into the tail vein using a 1 ml Sub-Qsyringe (BD).
Aerosol infection was performed as described elsewhere, using a Glas-col aerosol generator (BD) resulting in approximately 100 – 200 cfu of M. tuberculosis being deposited in the lung [page 42↓]of each mouse [57].

2.25. Determination of bacterial load


Infected mice were sacrificed at distinct time points by cervical dislocation. The examined organs were transferred to 1 ml PBS, 0.05 % Tween 80, homogenized and after serial dilution in PBS, 0.05 % Tween 80 plated on 7H11 agar plates supplemented with OADC (BD), cycloheximide (Merck), and ampicillin. After 3 – 4 weeks of incubation at 37°C the cfu were determined.

2.26. Histology


Tissue was fixed in 4 % formalin/PBS, dehydrated and embedded in paraffin, 5 µm sections were cut, and subsequently Hematoxylin and Eosin (H&E) stained. Rehydration was performed at 2 times 10 min Xylene, 2 x 5 min 95% ethanol, 2 x 5 min 80 % ethanol, 1 x 5 min 70 % ethanol, 1 x 5 min deionized H2O. For hematoxylin staining samples were incubated 1 x 10 min hematoxalin (Sigma), rinsed with water for 10 min, dipped in acid ethanol, rinsed in water, followed by Eosin Y (Sigma) staining for 1 min. Subsequently samples were rinsed in water and dehydrated for 3 min 95 % ethanol and 2 min Xylene. Samples were mounted using coverslip slides and permount.

2.27. Enzyme-Linked Immunosorbent Assay (ELISA)


Coating of 96 well plates was performed with 100 µl / well of monoclonal antibody rat anti-mouse IFNγ (clone R4-6A2, ATCC; 1 µg / ml) for 1 h at 37°C or over night at 4°C. Subsequently, 3 x washing with PBS 0.05 % Tween 20, 1 h blocking with 200 µl of blocking [page 43↓]buffer (PBS + 1 % BSA) for 1 h at 37°C and further 3 x washing. Standard (IFNγ [R&D] dilutions) and samples were added and incubated for 3 days. After 3 x washing, 1 h incubation with 100 µl XMG1.2-biotin second monoclonal antibody (1 µg / ml), and further 3 x washing, 100 µl of streptavidine alkaline phosphatase (Dianova, Hamburg, Germany) (diluted 1 : 2000) were added and incubated for 1 h at 37°C. Samples were 3 x washed, and 50 µl of substrate solution (1 tablet p-nitrophenyl-phosphate (Sigma) / 5ml di-ethanolamine-buffer (48.5 ml diethanolamine, 400 mg MgCl2, 100 mg NaN3 (0.02 %), ad 500 ml H2O, pH 9.8) per well was added. The reaction was stopped with 50 µl of 0.5 M EDTA, pH 8.0, and samples were measured at 405 nm / 490 nm.
IFNγ containing samples were collected from cells restimulated as follows: Cells were prepared from spleens which were passaged through a mesh, centrifuged for 5 min at 1500 rpm and 4°C. The pellet was resuspended in 1.5 ml erylyse-buffer.

erylyse-buffer:

8.29 g NH4Cl
1 g KHCO3
0.037 g EDTA
ad 1000 ml H2O and autoclaved

Subsequently, RPMI was added to a volume of 15 ml, the cells were centrifuged, resuspended in 10 ml RPMI, and adjusted to 1 x 106 / ml. One hundred µl were used per well and mixed with 100 µl medium or M. tuberculosis protein extract (10 µg / ml), respectively. After 3d of restimulation at 37°C / 5 % CO2, culture supernatants were collected and measured for IFNγ by ELISA.


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