by Carolyn Sholto-Douglas-Vernon

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department of Medical Biochemistry at the University of Stellenbosch

1 by Carolyn Sholto-Douglas-Vernon Thesis presented in partial fulfilment of the requirements for the degree ofdoctor of Philosophy in the department of Medical Biochemistry at the University of Stellenbosch April 2005 Supervisor: Professor P. van HeIden Co-supervisor: Professor T. Victor

2 DECLARATION I, the undersigned, hereby declare that the work contained in this thesis is my own original work and that I have not previously in its entirety or part submitted it at any university for a degree. Signature~':7?'" Date:...!4.:.P.4.~P.~ II

3 SUMMARY A gene coding for Arylaminie N-acetyltransferase (NAT) has been found in Mycobacterium tuberculosis, the casual agent oftuberculosis (TB). N-acetyltransferase acetylates and inactivates isoniazid (INH), which is a front line drug used in TB therapy. A guanine to adenine SNP at basepair 619 (G619A) has previously been identified in this gene, which results in a glycine to arginine change at amino acid 207 (G207R) (Upton et al. 2001). In this study the nat gene was further characterised. The frequency ofthe G619A SNP was analysed in 37 M tuberculosis strain families found in the Western Cape Province of South Africa, and it was found that the G619A SNP is conserved in two strain families (strain family 3 and strain family 28). Further sequence analysis identified a new thymine to cytosine SNP at base-pair 529 (T529C) resulting in a tyrosine to histidine change at amino acid 177 (Yl77H). This SNP was found only in isolates from strain family 3. These results imply that these SNPs may be used in epidemiology studies to classify isolates into these strain families. Using Real Time PCR, the expression of nat in M bovis BCG and M tuberculosis (reference strain H37Rv) was determined over a 7 and 28 day growth cycle, respectively. Using 16S rrna as an endogenous control, the nat gene was shown to be expressed early during the growth curve and reach its maximum expression level at approximately mid-log phase. The expression of nat was induced in drug susceptible M tuberculosis isolates (reference strain H37Rv and isolate 1430 containing both SNPs) exposed to INH at a concentration of O.Oll-lg/ml, but minimal change in expression was observed in resistant isolates (isolate 816) exposed to INH at the same concentration. Mycobacterium bovis BCG cultures exposed to INH, at a final concentration of 0.28I-lg/ml, showed an increase in protein production. The increase ofnat mrna and NAT protein in M tuberculosis and M bovis BCG, respectively, implies that INH affects the expression of NAT. The NAT protein was localised to all fractions ofthe cell in Mycobacterium smegmatis, M bovis BCG and M tuberculosis, using the Western blot technique. However, protein fractions from the cell envelope region showed a protein (detected with specific NAT antibodies) that ran at a higher molecular weight (MW). This implies that the cytosolic hydrophilic NAT undergoes some type of post-translational process that may make it hydrophobic, and enable it to pass into the cell envelope region. III

4 These results show for the first time how nat is expressed during the entire growth cycle of M tuberculosis and M. bovis BeG. It was shown that nat is expressed early during the growth cycle of the bacterium reaching maximum expression levels at mid-log phase. These results are in concordance with those obtained using M. smegmatis nat mutants, which taken together, show that early expression of nat is important for early growth and development ofmycobacteria. The results in this study also showed that NAT appeared to be translocated into the cell envelope of the bacterium, implying that NAT may be involved in one of the pathways needed for complete formation of the cell envelope. These results suggest that NAT may be an important target for drug development, as inhibitors ofnat could result in hindered growth and hence spread ofthe bacterium within its host. Inhibitors may also result in the incomplete development of the cell wall, enabling the host to combat the disease using its own immune system. IV

5 OPSOMMING 'n Geen wat kodeer vir Arielamien N-asetieltransferase (NAT), is gevind in Mycobacterium tuberculosis, die bacterium wat tuberkulose (TB) veroorsaak. NAT asetileer en inaktiveer isoniasied (INH), wat 'n belangrike antibiotika is in die behandeling van TB. 'n Enkel guanien na adenien nukleotied verandering (SNP) by basispaar 619 (G6l9A) is vroeer in hierdie geen gevind. Die basispaar verandering gee aanleiding tot 'n glisien na arginien aminosuur verandering by posisie 207 (G207R) in die proteyen (Upton et af. 2001). In hierdie studie is die nat geen verder gekarakteriseer. Die frekwensie van die G6l9A SNP is ondersoek in 37 verskillende M tuberculosis stamfamilie wat geisoleer was van TB pasiente in die Weskaap provinsie van Suid Afrika. Daar is gevind dat die G6l9A SNP teenwoordig is in twee stamfamilie (stamfamilie 3 en 28). Verdere DNA volgorde bepalings het 'n nuwe timien na sitosien SNP by basisparr 529 (T529C) getoon, wat aanleiding gee tot 'n tirosien na histidien aminosuur verandering by posisie 177 (Yl77H) in die proteyen. Hierdie SNP is slegs gevind in isolate van stamfamilie 3. Die resultate dui daarop dat hierdie SNP's gebruik kan word in epidimiologie studies om isolate van hierdie stamfamilies te identifiseer. Die uitdrukking van nat in M bovis BCG en M. tuberculosis (die verwysingsstam H37Rv is gebruik) is gedoen met "Real-Time PCR" na groeisiklusse van 7 en 28 dae van die bakterie, onderskeidilik. Die l6s rrna is as endogene kontrole gebruik om te wys dat die nat geen vroeg in die groeikurwe uitgedruk word en dat die maksimum uitdrukking verekry is by die middle eksponensiele van groei. Die uitdrukking van nat is geynduseer deur INH teen 'n konsentrasie van 0.01 Ilg/ml toe te dien in middlevatbare M tuberculosis isolate (H37Rv en isolaat 1430 wat albei SNP's bevat). 'n Minimale verandering in uitdrukking is gevind in weerstandige isolate (isolaat 816) wat blootgestel is aan INH. M. bovis BeG wat blootgestel was aan 'n finale konsentrasie van 0.28 Ilg/ml het a toename van proteyen produksie getoon. Die toename van nat mrna in M tuberculosis en NAT proteyen in M bovis BeG impliseer dat INH die uitdrukking van NAT affekteer. n' "Western blot" tegniek is gebruik om te wys dat die NAT proteyen gevind is in aile sel fraksies van M. smegmatis, M. bovis BeG en M. tuberculosis. ProteYen fraksies van die selomhulsel (selwand, selmembraan en intermembraan spasies) het egter 'n proteyen van 'n hoer moleklere gewig getoon (gevind deur NAT-spesifieke antiliggaampies te gebruik). Dit mag impliseer dat die v

6 hidrofiliese NAT proteren in die sitosol 'n na-translasie proses ondergaan wat die proteren hidrofobies maak sodat dit na die selomhulsel kan beweeg. Met hierdie resultate word vir die eerste keer gewys hoe die nat geen gedurende die hele groei siklus van M tuberculosis en M. bovis BeG uitgedruk word. Daar is gewys dat nat vroeg in die groeisiklus van die bacterium uitgedruk word en die hoogste vlakke word bereik in die middle eksponensiele groei fase. Hierdie resultate stem ooreen met wat verkry is wanneer M smegmatis nat-mutante gebruik is en dit dui daarop dat vroe uitdrukking van nat belangrik is vir groei en ontwikkling van mycobakterie. Die resultate wys ook dat NAT betrokke is by een van die paaie wat benodig word vir voledige vorming van die selomhulsel. Resultate van hierdie studie dui daarop dat NAT 'n belangrike teiken virmedikasie ontwikkeling kan wees, want inhibeerders mag veroorsaak dat die selwand onvolledig ontwikkel, wat die gasheer sal help om die bacterium dan met sy imuunsisteem te beveg. VI

7 ACKNOWLEDGEMENT I would like to acknowledge: My two supervisors at the University of Stellenbosch, Prof. P. van Heiden and Prof. T. Victor, for their patience, understanding and encouragement throughout this project. Prof. van HeIden for giving me the opportunity to do some ofmy research at the University ofoxford, Department ofpharmacology. Prof. E. Sim for allowing me to spend time within her lab at the University ofoxford, Department ofpharmacolgy. Prof. E. Sim for all her help and guidance during my stay in Oxford. All my colleagues both at Stellenbosch and Oxford for their continual encouragement and willingness to help whenever the need arose. VII

8 ABBREVIATIONS CI (3 'Y DC > < alpha beta gamma degrees Celsius greater than less than microlitre microgram micromolar number % 3D 1r Ax AcCoA ACP ADC AG agpl AIDs Arg Asp Ave BCG bp BSA cdna CFU C T Cys ddhzo percentage three dimensional pi absorbance at the wavelength ofx nanometres Acetyl Coenzyme A enoyl-acyl Carrier Protein Albumin-Dextrose Complex Arabinogalactan apolar glycopeptidolipid Acquired Immunodeficiency Syndrome Arginine Aspartate Average Bovine Calmette-Guerin base pair Bovine Serum Albumin copy Deoxynucleic Acid Colony Forming Unit threshold cycle Cysteine double distilled water VIII

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ENZYME PROFILING OF A RANGE OF SUGARCANE TISSUE TYPES WITH DIFFERENT LEVELS OF SUCROSE

ENZYME PROFILING OF A RANGE OF SUGARCANE TISSUE TYPES WITH DIFFERENT LEVELS OF SUCROSE by R. ORENDO-SMITH Thesis Presented for the degree Master of Science (Plant Biotechnology) at the University of Stellenbosch