Die Familie der Mykobakterien setzt sich aus pathogenen und apathogenen Vertretern zusammen. In dieser Arbeit wurden 3 Mitglieder dieser Familie für Untersuchungen herangezogen: ihr prominentester pathogener Vertreter Mycobacterium tuberculosis, der Erreger der Tuberkulose, das als Impfstoff eingesetzte Mycobacterium bovis BCG, das durch Attenuierung aus dem Rindertuberkulose-Erreger Mycobacterium bovis hervorging und das apathogene Bodenbakterium Mycobacterium smegmatis.
Ein Schlüssel zum Verständnis der Mykobakterien und speziell ihrer Widerstandsfähigkeit ist die Kenntnis ihrer komplexen Zellwand. Peptidoglycan als deren Bestandteil und insbesondere der mittels Undecaprenyl-Monophosphat bewerkstelligte Transport von Peptidoglycan-Vorläufern aus dem Cytoplasma an die Zelloberfläche steht dabei im Zentrum der Zellwandbildung. In M. tuberculosis, M. bovis BCG und M. smegmatis wurden Deletionsmutanten für die Undecaprenyl-Phosphokinase (Upk) hergestellt.
Für M. smegmatis wurde gezeigt, daß die Δupk Deletionsmutante, in Übereinstimmung mit Deletionsmutanten homologer Gene in anderen Bakterien, eine erhöhte Sensitivität gegenüber dem die Zellwandsynthese hemmenden Antibiotikum Bacitracin aufwies. Überraschenderweise zeigte M. tuberculosis Δupk diesen Phänotyp nicht. Weiterhin ließ sich für M. smegmatisΔupk im Vergleich zum M. smegmatis Wildtyp Peptidoglycan an der Zelloberfläche in geringerem Maße nachweisen. Eindrucksvoll zeigte sich die Bedeutung der Undecaprenyl Phosphokinase in der gestörten Entwicklung von Biofilmen im Falle der M. smegmatis
Δupk Mutante. Dies galt sowohl für in vitro Bedingungen als auch für ein, im Rahmen dieser Arbeit, neu entwickeltes in vivo Modell.
Vergleiche von M. tuberculosis Wildtyp und M. tuberculosis
Mutante auf der Ebene von Proteom- und Transkriptom-Analysen führten zur Identifikation eines zum mykobakteriellen Fettsäure-Synthese II (FASII) System gehörenden Operons, das im Falle der upk-Deletion verstärkt exprimiert wurde und damit möglicherweise einen Kompensationsmechanismus für die fehlende Phosphokinase darstellt.
Eine reduzierte Persistenz von M. smegmatisΔupk in infizierten Makrophagen legte nahe, daß Upk bei mykobakteriellen Infektionen eine entscheidende Rolle für das Überleben der Bakterien und ihre Virulenz spielt. Dies konnte erstmals für M. tuberculosis im Rahmen von Maus-Infektionsversuchen gezeigt werden. M. tuberculosisΔupk ließ sich als neues Mitglied in eine Reihe von als growth in vivo (giv) klassifizierten Mutanten einreihen.
Die Herstellung von Deletionsmutanten wird als Möglichkeit betrachtet, verbesserte Impfstoffe herzustellen. Die physiologische Konsequenz der Deletion sollte bestenfalls neben einer Attenuierung des Ausgangsbakteriums (gilt besonders für M. tuberculosis) eine Überexpression protektionsrelevanter Antigene zur Folge haben. Im Vergleich zum bestehenden Impfstoff M. bovis BCG führte die Impfung von Mäusen mit M. bovis BCG Δupk sowohl zu geringerer bakterieller im Anschluß an die Vakzinierung als auch zu einer verbesserten Langzeit-Protektion gegen Tuberkulose.
The family of mycobacteria is composed of pathogenic and apathogenic bacteria. This study was performed with 3 members of this family, the most prominent pathogenic member, Mycobacterium tuberculosis, the causative agent of tuberculosis, the vaccine strain Mycobacterium bovis BCG which was developed by attenuation of the bovine tuberculosis agent Mycobacterium bovis, and Mycobacterium smegmatis which is apathogenic and widely distributed in soil.
A key to understanding mycobacteria and, especially, their resistance is to understand the complexity of their cell wall. Peptidoglycan is a major component of the cell wall and the transport of peptidoglycan precursors out of the cytoplasm to the bacterial surface by undecaprenyl monophosphate is central to cell wall synthesis. Therefore, deletion mutants of the undecaprenyl phosphokinase gene (upk) were generated in M. tuberculosis, M. bovis BCG, and M. smegmatis.
In the case of M. smegmatis it was shown that a Δupk deletion mutant, as with deletion mutants of homologous genes in other bacteria, exhibited an increased sensitivity to the antibiotic bacitracin, indicating that cell wall synthesis was hampered. Surprisingly, M. tuberculosisΔupk did not exhibit this phenotype. Furthermore, a lower level of peptidoglycan was detected on the cell surface ofan M.
smegmatis Δupk mutant compared to M. smegmatis wildtype. Relevance of the undecaprenyl phosphokinase was demonstrated by impaired biofilm development in the case of the M. smegmatisΔupk mutant. This was observed in vitro as well as in vivo using an animal model which was newly developed in this thesis.
A fatty acid synthase II (FASII) system related operon revealed by comparative proteome- and transcriptome-analyses comparing M. tuberculosis wildtype and M. tuberculosisΔupk mutant, and may reflect a compensatory mechanism for the loss of upk.
Reduced persistence of M. smegmatis in infected macrophages suggested a decisive role of Upk in mycobacterial infection concerning survival and virulence of bacteria. This was later demonstrated to be true for M. tuberculosis in a mouse model. M. tuberculosisΔupk was, therefore, classified as a new member of the group of growth in vivo (giv) mutants.
Construction of deletion mutants is a strategy to identify improved vaccines. Ideally, the physiologic consequences of a gene deletion would result in attenuation of the modified bacterium (especially in the case of M. tuberculosis) and overexpression of antigens relevant for protection. Compared to the existing vaccine M. bovis BCG, vaccination of mice with M. bovis BCG Δupk exhibited a lower bacterial load upon vaccination as well as an improved long-lasting protection against M. tuberculosis
infection.
The family of mycobacteria comprises pathogens and apathogenic environmental bacteria [1,2]. Mycobacteria are unusual among bacteria since they have an enormously thick, hydrophobic cell wall which e.g. prevents desiccation. Numerous mycobacteria are harmless and useful because they degrade organic matter in soil. Better known are, however, the few human pathogenic mycobacteria which cause tuberculosis (Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis) and leprosy (Mycobacterium leprae). Discovery of M. tuberculosis
as an etiologic agent was reported to the “Physiologische Gesellschaft” in Berlin on March 24th, 1882 by Robert Koch. During his lecture on the “Ätiologie der Tuberkulose” Koch pointed out that it was a specific staining procedure that visualized characteristic, so far unknown bacteria in tuberculous affected organs [3]. The staining was performed with alkaline methylene blue and bismarck brown. Hence, M. tuberculosis as causal agent of tuberculosis was identified. At about the same time of Robert Koch’s discovery, Paul Clemens von Baumgarten brightened bacilli with caustic soda in caesous tissue and saw the bacteria under the microscope. Baumgarten’s publication came out a few weeks after Robert Koch’s lecture in the Charité in Berlin[4]. The staining procedures were modified first, by Paul Ehrlich, and subsequently by Franz Ziehl and Adolf Neelsen, to the use of a mixture of acids and aniline dye, today known as “Ziehl-Neelsen-staining” [5,6,7]. Therefore, the mycobacterial cell wall and its staining attributes were of notable importance right from the beginning.
From a public health standpoint, M. tuberculosis is of utmost importance because it kills more human beings than any other bacterial species
Robert Koch’s lecture on the “ Ätiologie der Tuberkulose ” provided an experimental methodology and an intellectual basis for acquiring knowledge about the cause of tuberculosis. Koch’s postulate was of great importance:
To prove that tuberculosis was caused by the invasion, growth and multiplication of the bacilli, it was necessary to:
grow them in pure culture, and
by administering the isolated bacilli to animals, reproduce similar moribund conditions.
Similarly, the knowledge of the molecular bases (the genotypes) for the phenotypes which allow M. tuberculosis to cause disease and escape therapies is essential in developing new effective treatment strategies. The conceptual methodology by which we acquire knowledge about what genotype is responsible for a particular phenotype is called Koch’s molecular postulate [8]:
To prove that a phenotype in a mutant bacterium, such as drug resistance or virulence, is caused by a specific genotype, it is necessary to:
Isolate a mutant with a defined altered phenotype,
clone the genotype from the mutant, and
by introducing the cloned genotype into a wildtype bacterium, reproduce the same phenotype of the mutant bacterium.
Based on this principle, a large number of genetic functional analyses regarding a variety of virulence and other factors in different bacteria have been achieved. Technological progress paved the way for the generation of mutant libraries and targeted gene deletions, as well as overproduction of selected genes. M. tuberculosis has been one of the most challenging and refractory organisms in this process. Nevertheless, there exists a set of tools to manipulate these bacilli [9], e.g. the
Soon after Robert Koch’s discovery of M. tuberculosis, innumerable attempts toward vaccine development began. On December 28th, 1908, the French bacteriologists Albert C. Calmette and Guérin notified a loss of virulence of M. bovis when cultured in bile containing media. These scientists passaged M. bovis over a period of 13 years in a bile-glycerin-medium thereby developing an attenuated strain that, when used as a vaccine, provided protection to high risk groups, especially newborns of tuberculous mothers. This vaccine strain was called Mycobacterium bovis BCG. It was introduced into the clinic in 1921 and is one of the oldest applications of vaccination. Nevertheless, there were severe complications [10] and later it was proven that M. bovis BCG fails to provide satisfying protection in adults against reactivated pulmonary tuberculosis [11,12]. In the Federal Republic of Germany the routine vaccination was discontinued in 1985 and has been offered since 1987 only upon request. Since there is no better anti-tuberculous vaccine than M. bovis BCG available so far, it still is the golden standard to which new candidates have to be compared in animal models.
The immune system is divided into two categories: innate and adaptive. Innate immunity refers to nonspecific defense mechanisms that come into play immediately or within hours of an antigen’s appearance in the body. These mechanisms include physical barriers such as skin, effector molecules in body fluids such as mucosal
Tuberculosis is caused by airborne infection. Inhaled droplets containing low numbers of bacteria are taken up by alveolar macrophages. Spontaneous healing cannot be measured nor excluded but is unlikely to occur to a significant degree. The vast majority, about 90 % of infected individuals, does not develop acute disease and stays latently infected. Patients with a compromised immune system, for example in the case of HIV infection, develop acute disease directly after primary infection [13]. Within 1 year more than 10 % of infected individuals develop disease. M. tuberculosis resides in early phagosomes and blocks phagosome maturation including phagolysosome formation [14,15,16,17]. However, the maturation arrest is incomplete and some bacteria are killed or at least impaired in replication through antibacterial effectors including reactive oxygen and nitrogen intermediates [18]. In addition, iron restriction is an important mechanism of innate immunity to control the infection [19].
The mycobacterial cell wall is impressively thick and unique in its complex composition [24]. Among the strategies by which M. tuberculosis has adapted to the environmental conditions in macrophages, the resistance imparted by its cell wall is one of the most striking. The cell envelope is extremely hydrophobic and forms an exceptionally strong permeability barrier, rendering mycobacteria naturally resistant to a wide variety of antimicrobial agents. This is due to the unique structure of the mycobacterial cell wall and the presence of long fatty acids, the mycolic acids [24]. Channel forming proteins which are functionally similar to the well known porins of gram negative bacteria have been demonstrated in Mycobacterium chelonae [25]
cytoplasm, membrane, and the cell wall itself (Fig. 3). Peptidoglycan synthesis is initiated with Uridine 5’-Diphospho-N-Acetylmuramic Acid (UDP-MurNAc) derived from Uridine 5’-Diphospho-DN-AcetylGlucosamine (UDP-GlcNAc) and phosphoenylpyruvate. Five amino acids are linked to UDP-MurNAc resulting in synthesis of UDP-MurNAc-pentapeptide, also known as Park’s nucleotide [29]. UDP
undecaprenyl-phosphokinase (Upk) probably represents a critical step in providing mycobacteria with a sufficient amount of peptidoglycan-precursor carrier molecules. Bacitracin is an antibiotic which binds tightly to C55-PP and blocks C55-P-recycling, in this way also reducing the amount of carrier molecules.
To study mycobacterial genetics of tuberculosis, the fast growing, environmental Mycobacterium smegmatis is widely used as a model organism. E.g., it was used to understand the effect of single M. tuberculosis genes on survival inside human macrophages [30]. Another potential use is to serve as surrogate for M. tuberculosis in disinfectant tests. Discovery of M. smegmatis was made 2 years after Robert Koch’s lecture on the “aetiology of tuberculosis”. In November 1884, Lustgarten was first to describe a bacillus with staining appearance of tubercle bacilli in syphilitic chancres and gummae [31]. Soon thereafter Alvarez and Tavel identified microorganisms similar to those in normal genital secretions (smegma) [32]. This kind of growth, as bacterial communities on a surface, is defined as a biofilm. Various gram-negative and gram-positive bacteria as well as fungi show two forms of growth: planktonic and, as a step of microbial development, in biofilms [33]. M. smegmatis and other non-tuberculous mycobacteria such as Mycobacterium fortuitum and Mycobacterium marinum live and grow planktonic or as biofilms [34].Biofilms support resistance to antimicrobial chemotherapy [35] and play a role in contamination of clinical and industrial settings [36]. M. smegmatis has also been described in wound infections, endocarditis, and osteomyelitis [37]. Nevertheless, it is considered as an apathogenic fast growing mycobacterium. Therefore, M. smegmatis
remains an attractive model organism to study mycobacterial metabolism or, specifically, cell wall synthesis as investigated in this study.
As mentioned, the cell envelope is a key to understanding mycobacteria and especially their resistance. Understanding initial processing, transport and assembly of peptidoglycan
1.) Isolate a mutant with a defined altered phenotype.
Attention was drawn to undecaprenyl phosphokinase, which is involved in peptidoglycan synthesis, and mediates resistance to the antibiotic bacitracin in the case of Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. Furthermore, the altered phenotype is characterized by decreased virulence for Staphylococcus aureus and Streptococcus pneumoniae [38,39].
2.) Clone the genotype from the mutant
The sequences of E. coli, S. aureus, and S. pneumoniae undecaprenyl phosphokinases are known. The homologous genes of M. tuberculosis, M. bovis BCG and M. smegmatis had to be identified, and appropriate knockout constructs had to be cloned.
3.) Introduce the cloned genotype into a wildtype bacterium, and reproduce the same phenotype of the mutant bacterium
.
Subsequently, the knockout constructs had to be used to introduce the deletions into the selected wildtype mycobacteria, followed by verification that a phenotype comparable to that
An additional aim was the more detailed characterization of Δupk mutants in different mycobacteria. This included determination of influence on peptidoglycan synthesis, bacitracin resistance, biofilm formation in the case of M. smegmatis, and virulence and persistence in the case of M. tuberculosis and M. bovis BCG. This was done in order to:
determine steps in cell wall synthesis which represent potential targets for drug development, and
generate an attenuated new vaccine candidate.
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).
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
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.
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
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.
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.
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
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:
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
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
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.
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
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
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
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
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.
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
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.
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.
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
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.
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)
ad 1000 ml
pH 9.8
sterile-filtered
Color change was visible after 1h at RT.
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
About 3 µl of a well-grown culture were transferred into subdivisions of a 8 chamber-slide
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.
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
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].
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.
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
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
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.
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.
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
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.
Protein sequences of E. coli undecaprenyl-phosphokinase (BacA), M. smegmatis undecaprenyl-phosphokinase (Upk), and M. tuberculosis Upk were compared by clustAl analysis (Fig. 6A). Both mycobacterial proteins showed 38 % identity to E. coli BacA and about 50 % similarity. ClustAl comparison of M. smegmatis and M. tuberculosis Upk revealed 73 % identity and 78 % similarity (Fig 6B). Highly conserved features were visible for all three homologues
An in-frame, unmarked deletion of the M. smegmatis
upk gene was created by a two-step-method [58]. Regions of 879 bp and 945 bp flanking the upk gene in M. smegmatis were amplified by PCR and inserted into pYUB657. Thus 96% of the wildtype upk gene was deleted. The mutated copy of upk was introduced into M. smegmatis by electroporation. HygR transformants which displayed a SucS phenotype were assayed for recombination by Southern blot. There is the possibility of integration upstream or downstream of the target gene by single crossover. A mutant in which the template plasmid has inserted upstream, resulting in 11,547 bp and 7,337 bp fragments on Southern blot (Fig 7., southern blot a) was chosen for further analysis. A culture of this single crossover strain was grown overnight in complete medium without hygromycin. This period of growth allowed double crossover events to occur, followed by plasmid loss. Deletion or preservation of the wildtype allele occur with equal probability. Two out of 6 analyzed clones displayed the characteristics of a upk deletion mutant (Fig. 7, southern blot b). The mutant strain was named M. smegmatisΔupk. A complementation strain was constructed, using pMV262-rv2136c, a multicopy plasmid expressing the M. tuberculosis H37Rv homologue of the upk
gene under control of the M. bovis BCG groEL2 (Hsp60) promoter. The parent plasmid, pMV262, is an episomal E. coli
-mycobacteria shuttle plasmid.
Bacteria were grown on 7H10 agar plates. While wildtype M. smegmatis showed dome-like morphology, Δupk mutant colonies exhibited caved-in structures (Fig. 8). Although this observation suggests that the cell wall is affected, examination by electron microscopy revealed no differences (Fig. 8). Yet, specific immunogold-staining of peptidoglycan revealed a lower number of gold-particles associated with the cell surface of the Δupk
mutant (Table 1). Twenty electron microscopy images of stainings of wildtype and the Δupk mutant were examined. For wildtype, 241 particles were counted of which 27% were surface associated. Of the 225 particles counted on the Δupk mutant bacteria, only 13% were surface associated (Fig. 9).
As described above, Δupk mutants are expected to be more sensitive to bacitracin than wildtype
M. smegmatis. This issue was investigated by the alamar blue assay. Alamar blue is a redox-dye that converts from blue to red upon reduction, reflecting a measure of growth [59]. Bacteria were incubated over night in 7H9 complete medium supplemented with serial dilutions of bacitracin (0 U/ml – 2,500 U/ml). Next day, alamar blue was added and fluorescence at 570 nm was measured. The M. smegmatisΔupk mutant showed no growth at a concentration of 500 U/ml bacitracin, whereas wildtype reached 55% of its maximal growth (Fig. 10). The reconstituted strain M. smegmatisΔupk + pMV262-rv2136c failed to regain wildtype growth but displayed an intermediate phenotype.
* Curves were significantly different at 500 and 1000 U bacitracin / ml according to Mann Whitney test (P<0.0001).
We wanted to find out whether upk homologues contribute to virulence of pathogenic mycobacteria. Therefore we chose the model of macrophage infection. Although, M. smegmatis fails to persist in macrophages it was possible to compare the survival times of M. smegmatis and the Δupk mutant in murine bone marrow derived macrophages. After infection, bacteria were cleared quickly. The time needed to kill half of the bacterial number upon infection differed between M. smegmatis wildtype (98 min), M. smegmatisΔupk mutant (27 min) and the reconstituted mutant strain (126 min) (Fig. 11).
Bacterial growth curves exhibited slightly slower growth of the M. smegmatisΔupk mutant compared to M. smegmatis wildtype after 9 h in 7H9 complete medium (Fig. 12A) and after 9 and 24 h in biofilm medium (Fig. 12B). Nevertheless, after 24 h, in 7H9 complete medium and after 72 h in biofilm medium, growth characteristics of all M. smegmatis strains adapted.
According to Mann Whitney test, growth curves in 7H9 complete medium (A) were not different whereas growth of M. smegmatis wildtype was significantly different at 9 and 24 h in biofilm medium(B) compared to the M. smegmatisΔupk strain and the reconstituted strain (P<0.0001).
Principally, an intact biofilm is characterized by a matrix consisting of extracellular polymer substances (EPS). Such a matrix was clearly visible for wildtype M. smegmatis, whereas the Δupk
mutant grew in separated single cells (Fig. 13). Additional studies to follow in vivo
The M. smegmatis upk gene was identified and an in-frame, unmarked deletion mutant was generated. The absence of Upk influenced bacitracin sensitivity, peptidoglycan synthesis, survival in macrophages, growth properties, and biofilm formation in vitro and in vivo. In vitro biofilms of M. smegmatis Δupk mutant were characterized as scattered islands of bacteria compared to a completely covered surface observed for wildtype bacteria. The M. smegmatis Δupk strain is the first to express a phenotype in a newly developed in vivo mouse model of biofilms.
A temperature sensitive (ts) TM4 phage delivery system was used to generate hygromycin-marked knockout mutants in M. tuberculosis and M. bovis BCG. Flanking regions to the M. tuberculosis upk gene were cloned next to a hygromycin resistance cassette and fused to the phage genome. Recombinant ts phages were amplified at 30°C using M. smegmatis growing in top agars as a host. At 37°C, phages were impaired to become lytic and could be used to transduce M. tuberculosis H37Rv and M. bovis BCG. The recombinant phages transferred their DNA into the bacteria. Flanking regions induced double crossover events, thus exchanging the upk gene with a hygromycin resistance cassette, which subsequently was verified by Southern blot (Fig. 15). The mutant strains were named:
M. tuberculosisΔupk and M. bovis BCG Δupk.
A complementation strain of M. tuberculosis was constructed using pMV262-rv2136c, a multicopy plasmid expressing upk
under control of the groEL2 (Hsp60) promoter of
M. bovis. The parent plasmid, pMV262, is an episomal E. coli
-mycobacteria shuttle-plasmid. M.
Growth properties in 7H9 medium were examined for the 3 strains: wildtype, Δupk mutant, and reconstitution. OD600 was measured once a day. The M. tuberculosisΔupk
mutant showed no differences in growth rate compared to wildtype whereas the complementation strain grew more slowly (Fig. 17).
Colonies of M. tuberculosis wildtype, the M. tuberculosisΔupk mutant and the reconstituted strain were of the same shape (data not shown). The attenuated strain M. tuberculosis H37Ra is known to behave differently compared to M. tuberculosis H37Rv in the following in vitro assays: Neutral red staining, cording assay, and pellicle culture. The H37Ra-strain is not stainable with neutral-red [61] (Fig. 18A).
For virulent M. tuberculosis H37Rv, bacterial cord-factors e.g. trehalose dimycolate (TDM; [62] are necessary for growth in cords on a surface in non-shaking cultures [63].
After 2 to 3 weeks, cultures of M. tuberculosis grown without shaking in Sauton medium, form pellicles on top of the medium and bacteria push each other up the wall of the tube.
All M. tuberculosis strains were examined in these 3 assays:
The strains were treated with neutral red. Pellets of stained Δupk
knockout mutant and complementation strain exhibited red color like H37Rv wildtype did, thus revealing no phenotype-differences (Fig. 18A).
Also cording was not affected by loss of upk. All strains were stained with rhodamine auramine (Fig. 18B).
The M. tuberculosis strains revealed phenotype-differences in case of pellicle formation. The Δupk mutant was hampered in pellicle formation and the complementation strain developed a
Cultures of M. tuberculosis H37Rv and the Δupk mutant were grown to late log-phase (OD600 = 1.0) and whole cell lysates, as well as RNA were purified.
Proteome analysis was performed with 3 independent protein lysates of both strains, M. tuberculosis wildtype and M. tuberculosisΔupk mutant. Each sample was analyzed on 2 gels. The comparative proteome analysis of 12 gels (6 wildtype and 6 mutant) revealed 45 spots of differential relative intensity. For the Δupk mutant, 23 spots belonged to the group of higher intensity and 22 spots exhibited lower intensity (Table 2) compared to M. tuberculosis wildtype. Representative examples are depicted in Figure 19. The 2-DE gels were first evaluated visually. Subsequently, the gels were re-evaluated by means of the image analysis program PDQuest. This software allowed verification of significance of the results by t-test and offered the possibility to quantify spot intensities.
In parallel to determine and compare gene expression profiles, RNA of M. tuberculosis H37Rv and the Δupk strain were purified and hybridized to a M. tuberculosis array. Wildtype and mutant showed a high number of differentially regulated genes. The 20 most significantly regulated genes of 2 independent experiments are summarized in Figures 20 and 21.
Correlation of the results of proteome and transcriptome was limited. Comparing M. tuberculosisΔupk and M. tuberculosis wildtype, proteins of higher spot intensity, such as
Thirty percent of the genes with a higher transcription rate in M. tuberculosisΔupk mutant compared to M. tuberculosis wildtype encode for proteins involved in lipid metabolism, additional 10 % were cell wall and cell-process related genes. Taken together, 40 % of the most significantly up-regulated genes were involved in the cell envelope complex. This corresponds to the observation that within the group of higher intensity protein spots, 37 %
Twenty percent of the genes with a lower transcription rate in M. tuberculosisΔupk encode for proteins which are related to the cell envelope complex. Again, this corresponds to 21 % of the protein spots with lower intensity which are related to cell envelope complex.
The alamar blue assay was used to determine sensitivity to antibiotics in the same way as described for M. smegmatis. Since there was no access to an Elisa-reader under biosafety level 3 (BSL3) conditions, completed assays were documented with a digital camera and visual color change was the value of bacterial growth. Alamar blue turns from blue to red upon reduction, indicating growth. Based on observations with M. smegmatis, the Δupk mutant was expected to be more sensitive to bacitracin. In contrast, no difference between M. tuberculosis H37Rv wildtype and Δupk mutant was detectable. The reconstituted strain was slightly more susceptible to bacitracin at minute concentration differences of the antibiotic. M. tuberculosis H37Rv and M. tuberculosisΔupk were resistant to bacitracin up to a concentration of 20 units / ml (Fig. 20A).
KasA and KasB which are over-expressed in the M. tuberculosisΔupk mutant, had been proposed to contribute to Isoniazid-resistance [66,67]. Sensitivity of the Δupk mutant to this antibiotic was therefore determined. All three examined M. tuberculosis strains were able to grow at concentrations of up to 20 µg Isoniazid / ml (Fig. 22) except for M. tuberculosisΔupk mutant which was slightly more susceptible, rather contradicting profound contribution of KasA to Isoniazid resistance.
To determine whether the upk deletion influenced growth of the mutant in vivo, C57BL/6 mice were infected intranasally with 1x103 cfu. Five mice per group, infected with M. tuberculosis H37Rv, M. tuberculosisΔupk, and M. tuberculosisΔupk + pMV262-upk, were sacrificed at day 1 to precisely determine the concentration of the inoculum. At day 30, 60, and 90, bacterial load in lung and spleen were determined (Fig. 23). M. tuberculosis H37Rv showed a characteristic growth curve with a plateau in the lung at 1x107 cfu. In contrast, cfu of the Δupk mutant increased less than a log per lung during the first 30 days and remained at a level of about 1.4 x 104 cfu / lung until day 90. The reconstituted strain reached an almost constant bacterial load of 1.8 x 103 cfu / lung at day 90. Nevertheless, all 3 strains were able to disseminate and were detected in spleen and liver (data not shown). While the burden of wildtype bacteria in spleen rose up to 1 x 105 cfu / organ by day 60, the burden of the upk mutant did exceed 4.1 x 103 cfu / spleen at day 30 and decreased afterwards. The phenotype of the reconstituted strain was unexpected and exhibited a slightly lower bacterial load than the M. tuberculosisΔupk strain in lung and spleen.
According to Mann Whitney test, bacterial numbers of M. tuberculosis wildtype and M. tuberculosisΔupk mutant were significantly different at day 60 and 90 in lung and spleen (P<0.0001).
Organs of infected animals, were removed for histological examinations. At day 90 post infection, lungs of M. tuberculosis wildtype and M. tuberculosisΔupk mutant infected mice revealed striking differences. Typically, animals infected with M. tuberculosis H37Rv showed severe pathology in the lung (Fig. 24). Granulomas were formed and a major part of the lung consisted of affected tissue. In the case of upk deficient M. tuberculosis (Fig. 25), granuloma and affected tissue were found but, in contrast to infection with M. tuberculosis wildtype, the majority of the lung appeared unaffected. The same observation was made for the reconstituted strain which failed to mimic the characteristics of the wildtype strain (Fig. 26).
Knowledge about virulence of a pathogen can be acquired by infection of immunocompromised mice which fail to control disease beyond a certain threshold. Commonly used immunocompromised animal models are i) highly susceptible Rag1-/- mice which lack T and B cells and hence, fail to mount an adaptive immune response [68] and ii) IFNγ-/- mice which are deficient in generating the central mediator of protection against tuberculosis. In this study, Rag1-/- mice were infected with 1x106 cfu mycobacteria. M. tuberculosis wildtype infected mice survived 26 days in the median (range: d23 - d49). Mice infected with M. tuberculosisΔupk survived 70 days in the median (range: d67 - d77) . Survival times of wildtype and mutant infected animals were significantly different according to logrank test (P<0.0001). The reconstituted strain killed mice after day 42 and about 50 % of the mice survived longer than those infected with the Δupk strain (Fig. 27A).
Similar to Rag1-/- mice, IFNγ-/- animals were less susceptible to the Δupk mutant strain than to M. tuberculosis H37Rv wildtype. The median survival time of wildtype infected animals was 30 days (range: d26 – d32), and for M. tuberculosis
Δupk mutant infected animals, 80 days (range: d63 – d126). Survival times of wildtype and mutant infected animals were significantly different according to logrank test (P<0.0001). Unexpectedly, the reconstituted strain was more attenuated than the mutant (Fig. 27B).
A upk deletion mutant (M. tuberculosisΔupk) was generated on a M. tuberculosis H37Rv background. In vitro growth properties of the mutant in shaking culture in 7H9 complete medium, cording, and neutral red to staining of this strain were unaffected. In addition, standing cultures in Sauton medium exhibited impaired pellicle formation. Global analysis of proteome and transcriptome revealed a high number of genes associated with lipid metabolism upregulated in the mutant, especially an operon of the mycobacterial FAS-II system. Increased sensitivity to bacitracin, however, was not detected. The upk deficient M. tuberculosis strain exhibited markedly reduced growth and pathology in vivo as well as reduced virulence in Rag1-/- and IFNγ-/- KO mice infections.
Balb/c mice were infected with 5 x 105 cfu of the M. bovis BCG Δupk strain and the M. bovis BCG wildtype strain i.v. and bacterial numbers were determined at days 15, 30, 60, and 90 post infection in lung and liver (Fig. 28). In the median, the bacterial burden in the lung of M. bovis BCG wildtype infected mice was about 2.17 times higher compared to mice infected with the upk deletion mutant, and 8.2 times higher in the liver. The small difference in bacterial load remained almost constant over the observed period of 90 days but was not significant according to “Mann Whitney” test (lung P = 0.5; liver P = 0.2).
IFNγ production by spleen cells of infected and of non infected mice was addressed by ELISA-assay (Fig. 29). Cells of whole spleens were either stimulated or left unstimulated with M. tuberculosis protein extracts. Spleen cells derived from naive mice failed to produce IFNγ, whilst cells of M. bovis BCG wildtype infected mice produced high levels of IFNγ at day 60 and day 90 post infection. Delayed IFNγ production of a comparably high level was measured at day 90 post infection in the case of spleen cells derived from mice infected with the BCG Δupk deletion mutant.
A major goal of studies with recombinant M. bovis BCG would be their utilization as improved vaccines. To exploit this possibility we compared vaccine efficacy of M. bovis BCG Δupk to the existing vaccine M. bovis BCG. Balb/c mice were vaccinated i.v. with 5 x 105 cfu. After 120 days these animals and an unvaccinated control group were challenged with about 200 cfu M. tuberculosis H37Rv delivered as aerosol. At distinct time-points thereafter, bacterial loads in lung and spleen were determined (Fig. 30). M. tuberculosis cfu differences in the spleen were very small between the groups 30, 60, and 120 days post infection. The M. tuberculosis burden in the lung of both groups of vaccinated animals was about the same and 60 fold lower compared to unvaccinated animals at day 30 post infection. However, by day 120 post infection, the BCG Δupk mutant strain was able to induce a significantly superior protection (Δlog = 2.7 in relation to naive mice) compared to wildtype M. bovis BCG (Δlog = 0.9) in the lung.
According to Mann Whitney test, bacterial numbers of M. bovis
BCG and the M. bovis
BCG Δupk mutant were significantly different at day 60 and 120 in the lung (P<0.0001).
Mice infected with the M. bovis BCG Δupk strain exhibited lower bacterial load in lung and liver compared to mice infected with wildtype BCG. As a consequence, a delayed IFNγ response was observed. Protection against M. tuberculosis challenge induced by the BCG Δupk mutant strain was highly improved in the lung of vaccinated animals at day 120 post infection compared to animals vaccinated with M. bovis BCG wildtype.
BacA is responsible for phosphorylation of undecaprenyl in E. coli and other bacteria. This is an important step in peptidoglycan synthesis. Studies on homologues of E. coli bacA in S. aureus and S. pneumoniae revealed that gene deletion mutants were attenuated in mouse models of infection [39]. We identified homologues in the genomes of M. smegmatis and M. tuberculosis (Upk; Fig. 1), and investigated the role of the cognate Upk in mycobacteriae, in vitro and in vivo. In addition, downstream consequences of the gene deletion in M. tuberculosis were investigated globally on the transcriptome and proteome level.
S. aureus and S. pneumoniaeΔbacA mutants show no significant alterations in growth rate or morphology [39]. In contrast, the in-frame, unmarked deletion of upk (Fig. 3), in M. smegmatis revealed a distinct phenotype (Fig. 4). While the growth rate was almost unaltered, M. smegmatis
Δupk colonies did not display dome-like structures, like that of the wildtype strain. Rather, they showed caved-in structures indicating autolysis of the cells. Upk deletion must be responsible for the altered colony morphology since complementation resulted in a return to dome-like colonies. A comparable phenomenon did not appear for M. tuberculosis or M. bovis BCG. This suggests that Upk may play different roles, or at least have different physiological functions, in different mycobacteria.
An altered cell wall might exhibit visible changes when examined by electron microscopy, however, the M. smegmatisΔupk cell wall appeared comparable to wildtype by electron microscopic examination (Fig. 8). More direct analyses of the cell wall by immuno-gold
As described for E. coli, S. aureus,and
S. pneumoniae, undecaprenyl phosphokinase deletion mutants exhibit higher sensitivity to bacitracin [39,38]. A knockout mutant strain of the mycobacterial homologue upk was expected to show the same susceptibility. This was verified unequivocally by the alamar blue assay for M. smegmatis (Fig. 10). Complementation with the M. tuberculosis upk homologue rv2136c partially reversed the susceptibility of M. smegmatis. Incomplete complementation may be due to constitutive gene expression of upk driven by the groEL2 (Hsp60) promoter, which does not reflect the physiological regulation of the gene. Surprisingly, the M. tuberculosis mutant strain was the first described bacterial upk deletion mutant that did not exhibit altered sensitivity to bacitracin. This finding demonstrates the uniqueness of the M. tuberculosis cell envelope not only to other bacteria but also to fast growing non-pathogenic species of mycobacteria [24,69], and emphasizes that even highly conserved proteins can have a range of activities in different species. M. tuberculosis may possess an efficient alternative pathway to shuttle out peptidoglycan precursors or may preserve the integrity of its cell wall by overproduction of other components. However, growth properties of M. tuberculosisΔupk in pellicle cultures were affected (Fig. 18) and this is likely due to altered surface features.
M. tuberculosis mouse infections never displayed a wildtype-like phenotype for M. tuberculosis Δupk + pMV262-upk. This phenomenon is likely due to weak transcriptional activity. For various genes the groEL2 promoter can provide the cell with more transcripts than the natural promoter, but for late log phase in vitro the groEL2 promoter transcribed about 200 fold weaker than the M. tuberculosis wildtype promoter. This severe regulation problem provides the most likely explanation for the failure of the complementation strain to achieve a wildtype phenotype in infection. In the case of M. tuberculosis pellicle formation, an intermediate phenotype of the complementation strain compared to wildtype and Δupk mutant was also observed.
Upk is thought to be critical for mycobacterial cell envelope formation and it was, therefore, expected that the Δupk mutant would attempt to compensate in some way. Global screening by proteome and transcriptome analysis was performed to identify putative compensatory mechanisms. One obvious response of the M. tuberculosisΔupk mutant was the upregulation of a FASII-system related operon (rv2243 – rv2247; Fig. 21). The involvement of KasA /
Mycobacterial FAS-II, unlike other bacterial type II FAS cognates, is incapable of de-novo fatty acid biosynthesis [73], however it appears able to elongate C14-AcpM of mycobacteria and C16-AcpM to preferentially long chain fatty acids ranging from 24 to 56 carbon atoms. Recent studies suggest that KasA (Rv2245) is part of FAS-II and participates in mycolic acid biosynthesis [74,75]. Mycolic acids are high molecular weight α-alkyl, β-hydroxy fatty acids with the general structure R-CH(OH)-CH(R’)-COOH, where R is a meromycolate chain consisting of 50 – 56 carbons and R’ is a shorter aliphatic branch possessing 22 – 26 carbons [24]. Mycolic acids are key components of the mycobacterial cell wall (Fig. 2) and play a role in producing an effective lipophilic barrier. Considering the importance of mycolic acids in bacterial survival and maintenance of cell wall integrity, the Δupk deletion mutant may
Infection of macrophages with M. smegmatis can be used as a model to analyze bacterial persistence in the host [30]. Lower persistence of the M. smegmatisΔupk mutant in macrophages indicates a role of Upk in mycobacterial virulence/persistence. Indeed, the M. smegmatisΔupk mutant was cleared more rapidly from the host cells. These findings indicate the importance of a robust cell envelope for persistence in the host. In this experiment complementation with the M. tuberculosis upk gene was sufficient to revert from mutant to wildtype phenotype. However, it is not valid to generalize knowledge gained from experiments with M. smegmatis. In certain aspects, M. tuberculosis is unique as was demonstrated in the case of resistance to bacitracin.
The term biofilm describes a population or community of bacteria living in organized structures at a liquid interface. Early confocal laser scanning microscopy (CLSM) of single species biofilms [79,80] revealed that biofilm bacteria live in cellular clusters or microcolonies that are encapsulated in a matrix composed of an extracellular polymeric substance (EPS), separated by open water channels which act as primitive circulatory system for the delivery of nutrients and removal of metabolic waste products. Within a biofilm, each bacterium occupies a specific microenvironment, which is defined by the surrounding cells, the proximity to a channel and the EPS matrix. The structuring of biofilms in microcolonies and fluid channels has been shown to be influenced by fluid flow, nutrient composition, and intracellular small messenger molecules, which are used for bacterial communication [81,82,83].
Various gram-negative and gram-positive bacteria, as well as fungi, grow in two forms: planktonic and, as a step of microbial development, in a biofilm [33]. M. smegmatis and other non-tuberculous mycobacteria such as Mycobacterium fortuitum and Mycobacterium marinum live and grow planktonicly or as a biofilm [34]. Biofilms support resistance to antimicrobial chemotherapy and play a role in contamination in clinical and industrial settings. Biofilm formation poses a major problem because they can increase drug resistance [36]. The reduced metabolic and growth rates shown by biofilm bacteria, particularly those deep within the biofilm, can render these microbes inherently less susceptible to antibiotics. The EPS matrix can act as an absorbent or reactant, thereby reducing the amount of drug available for action on biofilm cells. Moreover, biofilm bacteria are physiologically distinct from their planktonic cognates and express specific protective factors such as multidrug efflux
Biofilm growth of M. smegmatis was unaffected at Isoniazid concentrations that inhibited growth of planktonic bacilli [35]. Previously described deletion mutants of
M. smegmatis
lacking the capability of glycopeptidolipid acetylation, which affects the cell envelope, are defective in biofilm formation [86,60]. The in-frame, unmarked deletion mutant of the M. smegmatis upk gene is the first evidence for a role of Upk in biofilm formation. Upon adherence, the Δupk mutant strain formed a scattered biofilm only. Adherence could have been reduced due to a missing extracellular matrix (Fig. 13) and to slightly inferior growth-properties in biofilm medium.
The apathogenic environmental M. smegmatis owes its name from isolation from genital secretions (smegma): In November 1884, Lustgarten reported to the Royal Society of Medicine in Vienna that he had discovered a bacterium with staining characteristics of tubercle bacilli in syphilitic chancres and gummae [31]. Soon thereafter Alvarez and Tavel identified microorganisms similar to those in normal genital secretions (smegma) [32]. Smegma is associated with hygienic conditions and has been proposed as risk factor for penile cancer [87,88]. To determine whether Upk plays a role in genital smegma development by M. smegmatis we developed an in vivo model for M. smegmatis biofilm formation. In this in vivo mouse model of M. smegmatis biofilm formation the Δupk deletion mutant was found to be deficient in induction of smegma development (Fig. 14), thus stressing the relevance of Upk in mycobacterial saprophytic life.
A direct consequence of the upk gene deletion in M. tuberculosis
is the expression of a growth in vivo (giv) mutant phenotype. Studies on defined mutants of M. tuberculosis in the mouse model of infection have led to the classification of attenuated mutants in several phenotypic classes [89]. These mutants have been categorized by their growth characteristics, namely: i) severe growth in vivo (sgiv) mutants, which show a marked reduction in colony-forming units over time; ii) growth in vivo (giv) mutants, which grow less robustly than wildtype M. tuberculosis in the lungs of immunocompetent mice, yet still grow better than sgiv mutants; iii) persistence (per) mutants, which fail to grow or persist after the onset of acquired immunity, and iv) mutants with the same growth characteristics as per mutants, but show altered pathology (pat) compared with that of wildtype M. tuberculosis. Most mutants, including the Δupk mutant, fall into the giv class, showing reduced growth (Fig. 23) and pathology (Fig. 25), resulting in an attenuated phenotype and increased survival of infected immunocompromised mice (Fig. 27). Examples for giv mutants of M. tuberculosis
are the two component regulatory protein phoP [90], the accessory secretion factor secA2 [91], the glutamine synthase
glnA1
[92], and panCD [93], which is involved in pantothenate synthesis. The gene encoding the exported repetitive protein (erp) in both M. bovis BCG and M. tuberculosis has no ascribed function and is specific for mycobacterial species [94]. Deletion of the erp gene results in impaired growth of bacilli in lungs and spleens of infected mice, as with the Δupk mutant. Berthet et al. postulated that virulence depends on the ability of the bacilli to multiply [95]. The virulence reduced M. tuberculosisΔupk mutant exhibited characteristics of a giv mutant, which could render it an interesting vaccine candidate, because
The giv phenotype of the Δupk mutant, which was related to reduced growth in vivo, is probably related to its altered cell envelope. Forty percent of the most significantly upregulated genes in the Δupk mutant compared to wildtype were related to cell envelope processes and belonged to the tuberculist categories “lipid metabolism”, and “cell wall and cell processes” (Fig. 20). Hence, impaired self protection of the tubercle bacilli, as consequence of the impaired cell wall, or improved accessibility of antigens, or upregulation of one or more antigens which resulted in better processing and recognition by T cells, and therefore a more potent immune response, could allow improved control by the host. Furthermore, the M. tuberculosisΔupk mutant may switch prematurely to a dormancy program as may indicated by abundance of the HspX protein, a marker of M. tuberculosis latency [96,64].
In addition, host effector mechanisms may be more effective against the altered cell wall of M. tuberculosisΔupk than against M. tuberculosis wildtype. Such mechanisms may include production of highly reactive low molecular weight molecules, in particular reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) [97,98] (Fig. 1). ROI and RNI cause damage of cellular constituents by oxidation of cellular membranes and enzymes, DNA damage, mutagenesis, and inhibition of membrane transport processes [99,100,18].
Vaccine
As discussed above, the giv mutant phenotype of M. tuberculosisΔupk represents a promising phenotype for development of an attenuated M. tuberculosis mutant that could serve as a potential vaccine candidate. Nevertheless, a single gene deletion mutant in M. tuberculosis is unlikely to fulfill the safety standards required for a vaccine to be used in humans notably in immunocompromised individuals. Thus, a giv mutant like M. tuberculosisΔupk, needs to be thoroughly characterized and subsequently refined in further steps of development before it could become a valid vaccine candidate.
The vaccine strain M. bovis BCG offers an impressive safety record but unsatisfactory protection [101]. In this study, we deleted the upk gene from M. bovis BCG. This is an alternative strategy that relies on the basic premise that M. bovis BCG could be re-engineered to enhance its efficacy. For example, recombinant M. bovis BCG expressing various cytokines have been shown to improve the response against M. tuberculosis antigens [102] and recombinant M. bovis BCG expressing listeriolysin of Listeria monocytogenes showed an enhanced capacity to stimulate CD8+ T cells [103]. In addition, a recombinant M. bovis BCG
The upk deletion which resulted in an even more attenuated strain, was not expected to cause safety problems and accordingly used in a vaccine trial. Considering the lower bacterial load of M. bovis BCG Δupk upon vaccination (Fig. 28) and the delayed induction of the IFNγ response (Fig. 29) it was surprising that the modified vaccine strain was able to induce a significantly improved long-lasting protection against M. tuberculosis infection (Fig. 30). This phenotype is difficult to explain at present. Perhaps immunorelevant antigens were overexpressed to balance upk deficiency, or improved killing of M. bovis BCG Δupk provides the immune system with a larger amount of antigens to be processed, resulting in an improved adaptive immune response.
The mouse model of smegma was newly established and offers the opportunity for further development. The structure of smegmata can be investigated by microscopy and therefor allows the following questions to be addressed:
Is it possible to stain bacteria within the smegma?
Are the bacteria organized within the smegma?
As for biofilms, are channels existing for facilitated supply with nutrients?
Furthermore, the background growth which arose upon treatment, should be characterized to determine whether it contains other bacteria which induced genital secretions.
The reconstituted knockout strain of M. tuberculosis failed to exhibit the characteristics of a wildtype strain. Polar effects of the knockout construct may have disturbed transcription of downstream genes. It is also possible that the observed M. tuberculosisΔupk phenotype is due to disruption of the expression of an operon rather than a single gene. Transcription of putatively affected genes should be investigated by RT-PCR. And in the case of a disrupted operon, a reconstituted strain that complements all affected genes should be constructed.
It is still an unsolved problem why M. tuberculosisΔupk remains resistant to bacitracin.
The results derived from proteomic and transcriptomic analyses of M. tuberculosisΔupk strongly suggest an altered cell wall composition. An obvious experiment to follow this would be to extract lipids and to compare the differences between wildtype and mutant M. tuberculosis.
The promising results of the vaccine trial with M. bovis BCG Δupk advocate further development of the M. tuberculosisΔupk mutant strain. A M. tuberculosis mutant might induce a better protection against M. tuberculosis wildtype infections because it would prime the immune system with the same antigens as the causative agent M. tuberculosis, including antigens that are missing in M. bovis BCG like the 129 antigens of the 16 regions of deletion which were lost when Calmette and Guérin generated the vaccine strain from M. bovis. Nevertheless, for safety reasons, a one gene deletion mutant of M. tuberculosis will never be considered as vaccine candidate. Another phase of development is needed to further attenuate the strain and keep its protective potential at the same time. Nevertheless, the promising results of the vaccine trial with M. bovis BCG Δupk
suggest this strain has potential for future development as a vaccine candidate. Survival experiments with immunocompromised animals would be necessary to demonstrate its safety. In order to further investigate whether optimal protection is due to persistence of the vaccine strain, regulatory mechanisms of the immune system, or the timing/duration of IFNγ response upon vaccination, additional modified vaccine trials need to be performed.
Terness, P., Bauer, T. M., Röse, L., Dufter, C., Watzlik, A., Simon, H., and Opelz, G. (2002)
Inhibition of allogeneic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites. J.Exp.Med.
196, 447-457
Röse, L., Kaufmann, SHE., Daugelat, S
(2003)
Involvement of Mycobacterium smegmatis undecaprenyl phosphokinase in biofilm and smegma formation
manuscript status: submitted
Presentation of the poster:
Deletion of rv2136c in Mycobacterium tuberculosis.
on the Keystone symposium “Tuberculosis: Integrating Host and Pathogen Biology” was supported by the “Keystone Symposia Scholarship” (2003).
[1]
Mein Dank gilt Herrn Prof. Dr. Stefan Kaufmann für die Vergabe des Themas, die hervorragenden Arbeitsbedingungen, seine Bereitschaft zur wissenschaftlichen Diskussion und die mir gewährte Forschungsfreiheit während der Promotionsarbeit.
Frau Dr. Sabine Daugelat und Herrn Dr. Peter Seiler danke ich besonders für die exzellente Betreuung meiner Doktorarbeit, für zahlreiche fruchtbare wissenschaftliche Diskussionen und für viele hilfreiche Hinweise bei der Laborarbeit.
Herrn Dr. Jens Mattow sowie Eva Müller und Frank Siejak danke ich für die hervorragende Hilfe bei der Proteom-Analyse und Herrn Dr. Helmy Rachman für die Kooperation bei der Transkriptom-Analyse. Herrn Dr. Volker Brinkmann sei für die praktische Hilfe bei der Elektronen Mikroskopie gedankt.
Herrn Prof. Dr. Erwin Schneider gilt mein besonderer Dank für die Vertretung meiner Dissertation am Institut für Biologie der Humboldt-Universität zu Berlin und die Begutachtung der Arbeit. Ebenso danke ich Herrn PD Dr. Ulrich Schaible für seine Bereitschaft, die Arbeit zu begutachten. Bei Herrn Prof. Dr. Richard Lucius möchte ich mich für die Leitung des Promotionsausschusses bedanken. Schließlich bedanke ich mich bei Herrn Prof. Dr. Rainer Borriss dafür, daß er als Mitglied des Promotionsausschusses zur Verfügung stand.
Allen jetzigen und ehemaligen Mitgliedern des Max-Planck-Instituts für Infektionsbiologie, Abteilung Immunologie, gilt ebenfalls mein Dank für viele wichtige Anregungen und die angenehme Atmosphäre – auch außerhalb des Labors.
Meiner Freundin Nuran Özdugan danke ich für ihre liebevolle und geduldige Unterstützung. Schließlich möchte ich meinen Eltern für ihre ständige Zuneigung danken sowie dafür, daß Sie meine Ausbildung ermöglicht haben.
Hiermit erkläre ich, daß ich die vorliegende Arbeit selbständig und nur mit den angegebenen Hilfsmitteln erstellt habe.
Tables of strains, plasmids, and primers: