1.  Introduction

• 1.1. Koch’s postulate and Koch’s molecular postulate

• 1.2.  Mycobacterium bovis BCG

• 1.3. Immune response to M. tuberculosis

• 1.4. The mycobacterial cell wall

• 1.5.  Mycobacterium smegmatis

• 1.6. Aim of the study

2. Materials and Methods

• 2.1. Bacterial strains and culture methods

• 2.2.  Construction of the M. smegmatis knockout template

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

• 2.4. Alamar blue assay

• 2.5. Biofilm formation

• 2.6.  Infection of bone marrow derived mouse macrophages

• 2.7. Construction of a recombinant TM4 knockout phage

• 2.8. Transduction of M. tuberculosis

• 2.9.  Reconstitution of the mutants

• 2.10. Preparation of competent E. coli

• 2.11. Purification of chromosomal DNA from mycobacteriophages

• 2.12.  In vitro packaging of phasmid DNA

• 2.13.  Preparation of transduction competent E. coli HB101

• 2.14. Classical miniprep

• 2.15. Preparation of electro-competent mycobacteria

• 2.16. Neutral-red staining

• 2.17. Pellicle formation

• 2.18. Cording assay

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

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

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

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

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

• 2.24. Infection procedures

• 2.25. Determination of bacterial load

• 2.26. Histology

• 2.27. Enzyme-Linked Immunosorbent Assay (ELISA)

3.  Results

• 3.1.  M. smegmatisΔupk

  • 3.1.1.  Sequence comparison of Upk homologues

  • 3.1.2.  Construction of Δ upk mutant of M. smegmatis mc2 155

  • 3.1.3.  Differential abundance of peptidoglycan and colony morphology

  • 3.1.4.  Sensitivity to bacitracin

  • 3.1.5.  Accelerated clearance from infected macrophages

  • 3.1.6.  Growth properties and biofilm formation

  • 3.1.7.  Summary M. smegmatis mc2155 Δ upk

• 3.2.  M. tuberculosisΔupk (and construction of Mycobacterium bovis BCG Δupk)

  • 3.2.1.  Construction of Δ upk mutant strains of M. tuberculosis and M. bovis BCG

  • 3.2.2.  Growth curve

  • 3.2.3.  In vitro assays

  • 3.2.4.  Proteome and transcriptome analysis

  • 3.2.5.  Evaluation of sensitivity to antibiotics

  • 3.2.6.  Infection studies

  • 3.2.7.  Histology

  • 3.2.8.  Survival

  • 3.2.9.  Summary M. tuberculosis H37Rv Δ upk

• 3.3.  M. bovis BCG Δupk

  • 3.3.1.  Infection studies

  • 3.3.2.  IFN γ production of stimulated spleen cells

  • 3.3.3.  Vaccine trial

  • 3.3.4.  Summary M. bovis BCG Δ upk

4.  Discussion

• 4.1. Impact of upk deletion on cell wall attributes

• 4.2. Physiological balance

• 4.3.  In vivo

• 4.4.  Outlook

5.  Publications

Appendix

Table 1: distribution of gold particles in anti peptidoglycan immuno-gold stain.

Table 2a: M. tuberculosis H37Rv Δ upk protein spots of higher relative intensity

Table 2b: M. tuberculosis H37Rv Δ upk protein spots of lower relative intensity

Table 3a: M. tuberculosis H37Rv Δ upk genes of higher transcription rate compared to wildtype

Table 3b: M. tuberculosis H37Rv Δ upk genes of lower transcription rate compared to wildtype

Table 4: functional categories

Table 4: Strains and plasmids used in this study

Table 5: List of primers

Fig. 1 Scheme of the course of events following contagion with M. tuberculosis. Acute disease does only develop for a relatively small subset of immunocompromised individuals. Depicted are the major effector mechanisms of macrophages and the most important T cell populations. (Reprinted from Nature Reviews Immunology [20]).

Fig. 2 Scheme of the mycobacterial cell wall structure. The mycolyl-arabinogalactan-peptidoglycan complex forms the core of the robust bacterial envelope.

Fig. 3 Model of peptidoglycan synthesis. Precursors are produced in the cytoplasm subsequently coupled to a undecaprenyl monophosphate, the carrier lipid, and modified. After translocation to the outer face, peptidoglycan is assembled and incorporated into the cell wall.

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.

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

Fig. 6 Protein sequence comparison with clustAl alignment reveals highly conserved features of E. coli BacA and mycobacterial Upk. Identity of E. coli to M. smegmatis and M. tuberculosis is 38 % and similarity 50 %(A). Identity of M. smegmatis to M. tuberculosis is 73 %, and similarity 78 %(B). Color definitions: blue - all amino acids of a column are identical; red - more than half of the amino acids of a column are identical or belong to one of the strong groups (amino acids with strong similarities); orange - more than half of the amino acids of a column belong to one of the weak groups (amino acids with weak similarities), or amino acids that could be grouped into a weak group with every amino acid of the same column belonging to a strong group that is marked red.

Fig. 7 An in-frame, unmarked deletion of upk was generated in M. smegmatis by a two step approach. In the first step, the counterselectable suicide plasmid pYUB657 carrying the deletion allele of upk recombined with the bacterial chromosome. Southern blot analysis confirmed the recombination event. Two orientations are possible (southern blot a: lane 1 and 2). Clone 2 was selected. In the second step the plasmid loops out in the absence of hygromycin selective pressure. The deletion allele or the wildtype copy of upk are lost with equal probability. Deletion of upk was verified by Southern blot (Southern blot b). Lane 1 and 5 are deletions, the others are wildtype. For the following studies clone number 1 was selected.

Fig. 8 Differential colony morphology of M. smegmatis mc2155 and M. smegmatis mc2155Δupk. Mutant colonies have caved-in structures lacking dense cores (1a: wildtype and 2a: Δupk mutant). Using electron microscopy, there is no visible difference in cell wall architecture (1: wildtype and 2: Δupk mutant).

Fig. 9 Distribution of gold particles in an anti-peptidoglycan immuno-gold stain. Twenty electron microscopy images 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, 13% were surface associated.

Fig. 10 Sensitivity assay to bacitracin. M. smegmatis mc2155 (■), M. smegmatis mc2155Δupk (□),and M. smegmatis mc2155Δupk + pMV262-rv2136c (●) were incubated with different concentrations of bacitracin over night. Next day alamar blue was added and growth was measured at 570 nm. The Figure shows 1 representative experiment of 3 with similar results. * Curves were significantly different at 500 and 1000 U bacitracin / ml according to Mann Whitney test (P<0.0001).

Fig. 11 Infection of murine bone marrow derived macrophages. M. smegmatis mc2155 (■), M. smegmatis mc2155Δupk (□) and M. smegmatis mc2155Δupk + pMV262-rv2136c (●) were used at a MOI of 200. After 27 min half of M. smegmatisΔupk mutants were killed. This was true for wildtype M. smegmatis after 98 min and after 126 min for Δupk mutant carrying pMV262-rv2136c. The Figure shows 1 representative experiment of 2 with similar results.

Fig. 12 Growth curves. M. smegmatis mc2155 (■), M. smegmatis mc2155Δupk (□), and M. smegmatis mc2155Δupk + pMV262-rv2136c (●) grown at 37°C in 7H9 complete medium (A) and biofilm medium (B). The Figure shows 1 representative experiment of 2 with similar results. 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).

Fig. 13 Differential biofilm formation. M. smegmatis mc2155 (A, C) and M. smegmatis mc2155Δupk (B, D) were grown for 4 to 5 days at room temperature in biofilm medium and stained with crystal violet. The mutant exhibited impaired biofilm development. Rhodamine auramine staining (C, D) visualized even structures (highlighted by white arrows) only in wildtype biofilms.

Fig. 14A In vivo biofilm formation. Penises of C57BL/6 mice were exposed to medium, wildtype M. smegmatis, and Δupk deletion mutant. The Figure shows combined results of 2 independent experiments with similar results. Before application of the M. smegmatis strains, mice had a clean penis (14B). A penis with an over night grown smegma is depicted in 14C. 14D shows a smegma plug removed from a penis.

Fig. 15 Construction of upk knockout mutants in M. tuberculosis and M. bovis BCG. Knockout phage genome phAE159-Δupk was generated by ligation of the plasmid pKO-upk and the phage genome phAE159 TM4 (A). Four phages were tested for temperature-sensitivity and revealed the expected phenotype- lysis at 30°C but not at 37°C (B). # 4 was picked, amplified and used for transduction of M. tuberculosis and M. bovis BCG. Lane 4 and 6 represent upk deletion mutants for M. tuberculosis H37Rv and M. bovis BCG, verified by Southern blot analysis (C).

Fig. 16 Comparison of the original reconstitution vector pMV262-rv2136c, and the reconstitution vector purified from an electroporated Δupk strain. Lanes # 1 and # 2 show the pattern of the original insert with accurate orientation. Lanes # 3 and # 4 confirm this pattern for the vector purified from the reconstitution strain.

Fig. 17 Growth curves. M. tuberculosis H37Rv (■), M. tuberculosis H37Rv Δupk (□), and M. tuberculosis H37Rv Δupk + pMV262-rv2136c (∙) were grown at 37°C in 7H9 complete medium. The Figure shows 1 representative experiment of 2 with similar results.

Fig. 18 Assays on M. tuberculosis behavior under defined conditions. Neutral-red stained M. tuberculosis H37Rv, M. tuberculosisΔupk, and the reconstitution strain but not M. tuberculosis H37Ra (A). Wildtype, mutant, and reconstitution strain grew in cords (B). M. tuberculosis wildtype formed a heterogeneous pellicle whereas M. tuberculosisΔupk was retarded in pellicle formation, and the reconstituted mutant exhibited a smooth pellicle (C)

Fig. 19 Examples of protein spots with differential relative intensity in 2-DE patterns of whole cell lysate proteins of the upk mutant strain (B, D, F) and the M. tuberculosis H37Rv control (A, C, E). The same protein can give rise to multiple spots as in the case of Rv2031c/HspX (picture E).

Fig. 20 Percentage distribution of gene expression profiles and differential protein spot intensities according totuberculist classification of functional categories. Lipid metabolism was the most prominent fraction within the group of up-regulated genes for the Δupk mutant (A) and insertion sequences and phages protrude as a cluster of genes to be turned off (B).

Fig. 21 Genomic organization of the Fas-II system related operon encoding enzymes involved in biosynthetic pathway for long-chain fatty acids. Genes / proteins with higher transcription rate / amount of protein in the upk deletion mutant are highlighted by arrows

Fig. 22 Alamar blue assay. M. tuberculosis H37Rv wildtype (H37Rv), Δupk mutant (Δupk), and the reconstituted mutant (Rec) were tested for sensitivity against bacitracin (A) and Isoniazid (B). Red color indicates growth / resistance, blue indicates no growth / sensitivity. The Figure shows representative experiments of 4 with similar results.

Fig. 23 Growth of M. tuberculosis wildtype, the upk deficient, and the reconstituted strain in lung and spleen. C57BL/6 mice were infected with 1x103 cfu. Filled squares represent cfu of wildtype M. tuberculosis, open squares M. tuberculosisΔupk deletion mutant, and filled reconstitution strain. The Figure shows 1 representative experiment of 2 with similar results. 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).

Fig. 24 Lungs of mice infected with M. tuberculosis H37Rv exhibited severe pathology 90 days post infection. Major part of the lung consisted of granulomatous, heavily infiltrated tissue. Only small regions with unaffected alveoli were found.

Fig. 25 Lungs of mice infected with M. tuberculosis H37Rv Δupk exhibited reduced pathology compared to M. tuberculosis wildtype 90 days post infection. Major part of the lung appeared non-infiltrated. Only small parts with granuloma could be detected.

Fig. 26 Lungs of mice infected with the reconstitution strain of M. tuberculosis H37Rv Δupk exhibited – like M. tuberculosisΔupk - reduced pathology compared to M. tuberculosis wildtype 90 days post infection. Major part of the lung appeared non-infiltrated. Only small parts with granuloma could be detected.

Fig. 27 Survival of immunocompromised mice. Rag1-/- (A) and IFNγ deficient animals (B) were infected with M. tuberculosis H37Rv(■), upk deficient M. tuberculosis (□), and the reconstituted strain (●), respectively. The experiments were performed with 10 mice per group. Survival of Rag1-/- deficient animals was determined once. Figure B shows 1 representative experiment of 2 with similar results.

Fig. 28 Growth of wildtype and upk-deficient M. bovis BCG in lung and liver of Balb/C mice infected with 5 x 105 CFU. Filled squares represent cfu of wildtype M. bovis BCG, and open squares represent cfu of the M. bovis BCG upk deletion mutant. At each time-point 5 mice per group were sacrificed.

Fig. 29 Measurement of IFNγ production by ELISA. Spleen cells of M. bovis BCG inoculated mice produced high levels of IFNγ upon stimulation at day 60 and 90 post infection. Within each group, left column displays unstimulated, right column displays stimulated samples. Spleen cells of mice vaccinated with upk deficient M. bovis BCG strain produced a delayed but also high response at day 90 post infection.

Fig. 30 Bacterial burden of Balb/c micechallenged with M. tuberculosis H37Rv viaaerosol. Filled squares represent M. tuberculosis cfu of wildtype M. bovis BCG vaccinated animals, open squares represent M. tuberculosis cfu of the M. bovis BCG Δupk vaccinated animals, crosses represent M. tuberculosis cfu of unvaccinated animals. 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).