Hospitals worldwide are becoming increasingly plagued by antibiotic-resistant pathogens; concomitantly, the number of patients who die from antibiotic-resistant pathogens is increasing. The rise of multi-drug resistant (MDR) pathogens has rendered many antibiotics obsolete. The streptogramin and aminoglycoside antibiotics are drugs of last resort against life-threatening, MDR Gram-positive (e.g., methicillin resistant Staphylococcus aureus (MRSA)) and -negative (e.g., Pseudomonas aeruginosa) pathogens, respectively; however, as microbial drug resistance continues to emerge, the efficacy of these two important drug classes is decreasing. One of the most common mechanisms by which pathogens become resistant to streptogramin and aminoglycoside antibiotics is enzymatic inactivation: the Vat and AAC(3) acetyltransferases are employed by pathogens to inactivate streptogramin and aminoglycoside antibiotics, respectively. There currently is a dire need to not only develop new antibiotics, but to find new, creative strategies to outwit microbial resistance mechanisms. One of these strategies is to rescue the activity of antibiotics through the discovery of antibiotic adjuvants. In the current study, adjuvants which rescue the activity of streptogramin and aminoglycoside antibiotics through inhibition of the resistance acetyltransferases, VatD and AAC(3)-Ia, have been discovered—through the development of a cell-based screening method, we have found the first inhibitors of VatD, as well as of AAC(3)-Ia and its homologues, AAC(3)-Ib, AAC(3)-Ic, and AAC(3)-Id. We have demonstrated that streptogramin and aminoglycoside resistance can be reversed both in vitro and in vivo by the protein kinase inhibitors, GW5074 and rottlerin. Steady state kinetics revealed these compounds to inhibit VatD and AAC(3) enzymes mainly through noncompetitive or mixed mechanisms. This study has also demonstrated that eukaryotic kinase inhibitor libraries may be repurposed for the discovery of not only new antibiotic adjuvants, but also new antimicrobial targets. The inhibitors described herein may someday serve as effective adjuvants of streptogramin and aminoglycoside antibiotics. / Thesis / Doctor of Philosophy (PhD)

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<P> The first histone acetyltransferase to be described was GCN5, from the yeast species Saccharomyces cerevisiae. To date, the GCN5-related N-acetyltransferases (GNATs) comprise one of the largest enzyme superfamilies with over 10,000 identified members in sequenced genomes. This protein is known to acetylate specific lysine residues on the amino-terminal tails of nucleosomal histones, thereby loosening their contact with the tightly packed DNA and facilitating transcription. </p> <p> In this study, I determined that GCN5 is able to shuttle between the nucleus and the cytoplasm using fluorescence recovery after photobleaching (FRAP). Mutational studies revealed that its nuclear import is regulated by a classical bipartite nuclear localization signal (NLS) that is dependent on the transporters importin a and f3. In contrast, we found that GCN5 lacks a CRM1-dependent nuclear export signal (NES), as demonstrated by mutational and leptomycin B (LMB) studies; instead, IKB, a previouslydescribed transcription inhibitor with a CRMl-dependent NES, was found to modulate the export of GCN5 from the nucleus. This was initially discovered while performing the LMB assays, for which IKB served as a positive control, and was subsequently confirmed by mutational studies and protein complementation assays (PCAs). Furthermore, while the PCAs demonstrated a physical interaction between these two proteins in vivo, GST pull-down experiments were employed to confirm their interaction in vitro. </p> <p> Furthermore, this study also revealed that over-expression of GCN5-e YFP in NIH 3T3 cells causes -10% of the transfected cells to exhibit nuclear GCN5-eYFP-associated filaments; these structures were confirmed to be F -actin filaments comprised of f3-actin through co-localization studies with both TRITC-phalloidin and a mRFP-f-actin construct. GCN5's acetyltransferase activity was shown to be responsible for the formation ofthese filaments through mutation of its catalytic residue. Moreover, a protein complementation assay (PCA) demonstrated an in vivo interaction between GCN5 and f-actin, while FRAP analysis of a single filament showed that GCN5-e YFP molecules rapidly and randomly associate with these filaments along their entire length. Together these results suggest that GCN5's acetyltransferase activity is responsible for the structural maintenance of these filaments. Finally, GCN5-eYFP-associated filaments were found to be spatially separate from both lamin A (a nuclear envelope structural protein) and DNA; however, this does not exclude the possibility of an indirect interaction between these cellular constituents, as treatment of a live cell with Hoechst DNA stain, which disrupts the structure of DNA, was shown to disturb the structural integrity of these filaments. </p> / Thesis / Master of Science (MSc)

Gegenstand der Untersuchung: Vorliegende Arbeit stellt eine molekularbiologische Studie dar, die der Frage nachging, ob der Genotyp für die NAT2 eine Rolle bei der Pathogenese des Mammakarzinoms spielt. Das Mammakarzinom hat eine erbliche Komponente. Neben hoch-penetranten genetischen Mutationen des BRCA1- und BRCA2-Gens stehen polymorphe Enzyme des Fremdstoffwechsels im Verdacht, Präkanzerogene zu aktivieren und somit das Karzinomrisiko zu erhöhen. Die NAT2 detoxifiziert aromatische Amine, wie sie z. B. im Zigarettenrauch enthalten sind und weist eine ausgesprochene bimodale Aktivitätsverteilung auf. Nach systematischer Aufklärung des genetischen Polymorphismus der NAT2 ist eine Vorhersage des Phänotyps mit hoher Sicherheit möglich. Design: Es wurde eine prospektive Fall-Kontroll-Studie an 248 Patientinnen mit Mammakarzinom und 248 Kontrollen (Patientinnen mit anderen, nicht malignen Erkrankungen und Gesunde) durchgeführt. Zusätzlich wurde eine Blutprobe entnommen, aus der DNA isoliert wurde. Damit wurden folgende Mutationen des NAT2-Gens bestimmt: G191A, C282T, T341C, C481T, G590A, A803G, G857A. Anhand dieser Mutationen erfolgte die Zuordnung zu Haplotypen. Da bekannt ist, welche mittlere Aktivität der NAT2 bei den einzelnen Genotypen zu erwarten ist, konnte mit dieser Information eine Vorhersage der Aktivität als "schnelle" und "langsame" Acetylierer vorgenommen werden. Methode: Die Genotypisierung erfolgte durch Amplifikation des NAT2 -Gens mit verschiedenen Primern in der PCR, anschließendem Verdau mit Restriktionsenzymen und Charakterisierung der Fragmente mittels Gelelektrophorese. Statistik: Mit der Berechnung von "Odds ratios" und multivariaten logistischen Regressionsanalysen zur Berücksichtigung möglicher Einflussfaktoren wurde der Zusammenhang zwischen NAT2-Genotyp bzw. daraus vorhergesagtem Phänotyp und Mammakarzinom überprüft. Ergebnisse: Es wurden acht verschiedene NAT2-Haplotypen nachgewiesen. 2 Haplotypen (*4 und *12A) kodieren für einen schnellen Acetylierer-Typ, 6 Haplotypen (5A, 5B, 5C, 6A, 7B und 14*B) für den langsamen Acetylierer-Typ. 55,6% der MC-Patientinnen und 58% der Kontrollpersonen Genotypen wiesen den langsamen Acetyliererstatus auf. Dies entspricht in etwa der von anderen Untersuchern beschriebenen Häufigkeit bei Kaukasiern. Es fand sich keine Überrepräsentierung bestimmter Genotypen oder Haplotypen in der Gesamtgruppe der Patientinnen mit Mammakarzinom im Vergleich zur Kontrollgruppe. Die Odds ratio betrug 1,12 (CI: 0,03 - 1,60). In einer Subgruppenanalyse fanden sich keine signifikante Unterschiede in bezug auf das mittlere Alter, das Ausmaß des Zigarettenkonsums und auch von Blutgruppenmerkmalen. Innerhalb des Patientenkollektivs wurde die Verteilung von schnellen und langsamen Acetylierern stratifiziert nach Menopausenstatus, Menstruationsdauer, TNM-Klassifikation und Grading. Bei den Patientinnen mit invasiv-lobulärer Tumorhistologie waren die schnellen Acetylierer in der Auswertung signifikant häufiger als bei invasiv-duktalem Mammakarzinom. Ein weiterer signifikanter Unterschied wird bezüglich des Hormonrezeptorstatus berichtet: Schnelle Acetylierer waren bei Patientinnen mit positivem Hormonrezeptorstatus deutlich häufiger als bei solchen mit negativem Rezeptorstatus. Bezüglich der Tumorhistologie fiel auf, dass die 33 Patientinnen mit einem invasiv-lobulären Mammakarzinom signifikant häufiger schnelle Acetylierer waren. Langsame Acetylierer wiesen dagegen häufiger einen negativen Östrogenrezeptorstatus auf. Schlussfolgerung: Dem Polymorphismus des NAT2-Genotyps als unabhängigem Risikofaktor bei der Entstehung des Mammakarzinoms kommt keine mit den bekannten Risikofaktoren vergleichbare Bedeutung zu. Die Befunde in bezug auf die Tumorhistologie und auf den Hormonrezeptorstatus weisen möglicherweise auf Besonderheiten im Pathomechanismus bei der Entstehung des Mammakarzinoms hin, der derzeit unklar ist. Sie bedürfen weiterer Abklärung. / Genetically polymorphic xenobiotic metabolizing enzymes such as the polymorphic arylamine N-acetyltransferase (NAT2) are supposed to be a host factor for cancer susceptibility. A case-control study of a total of 248 patients with breast cancer and a matched reference group of 248 unrelated subjects without cancer was performed to explore the association between NAT2 genetic polymorphism and individual susceptibility to breast cancer. A structured questionnaire was used to collect relevant information regarding all known or suspected risk factors of breast cancer. Methods: The NAT2 genotype was determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The NAT2-genotype was characterized at nucleotide positions 191, 282, 341, 481, 590, 803, and 857. For evaluation of nucleotide 341, a 3’-mismatch primer was used. Homozygous wild type genotypes NAT2*4/*4 were confirmed by DNA sequencing. Results: Genotypes for rapid acetylation amounted to 44.4% among breast cancer and 41.5% among reference patients. There was no over-representation of specific NAT2-genotypes in the total of breast cancer patients compared with the reference group (odds ratio 1.12, 95%; confidence limits 0.03-1.60). Neither NAT2-status nor smoking status was independently associated with breast cancer risk. Logistic regression analysis, considering confounders such as age, body mass index (BMI), and smoking status (PJ) showed that the NAT2 rapid acetylator genotype was not associated with an increased cancer risk (odds ratio 1.05; 95%-CI: 0.67-1.67; P=0.82). Discrimination into homozygous and heterozygous carriers of allele NAT2*4 did not show any over-representation of NAT2*4/*4 genotypes among breast cancer patients (odds ratio 1.31; 95% confidence limits 0.59-2.91; p=0.50). Hence carriers of the NAT2*4/*4 genotypes, with its especially high acetylation capacity are not at significantly increased risk to breast cancer. Further stratification to different risk factors revealed a non-significant elevation in risk of breast cancer among patients with increasing cigarette smoking who represented the NAT2 rapid acetylator genotype, but lack of association to age, blood groups, menopause, period of menstruation, TNM-classification, tumor grading, and histology. However, evaluation of the role of estrogen receptor status and NAT2 showed that there was a significant association between positive receptor status and NAT2 rapid acetylator genotype (odds ratio 2.07; 95%-CI: 1.32-5.27; P=0.005). Interestingly, in patients with infiltrating lobular breast cancer (n = 33), NAT2 rapid acetylator genotypes were more frequent compared with other tumor subtypes (odds ratio 2.59; 95%-CI: 1.20-5.60; P=0.014). Logistic regression analysis, considering estrogen receptor status, and age showed that the rapid acetylator genotypes were associated with an increased cancer risk (odds ratio 2.4; 95%-CI: 1.04-5.61; P=0.04). Our findings suggest that NAT2-polymorphism is not an independent susceptibility factor for breast cancer. In particular the NAT2 slow acetylator genotype was not associated with an increased breast cancer risk. Striking results point out to a likely association between NAT2 rapid acetylator genotype and tumor histology especially infiltrating lobular breast cancer and positive hormone receptor status These findings refer to special features of pathogenesis in breast cancer requiring more and detailed clarification.

Les complexes acétyltransférases (HAT), SAGA et ATAC, sont des régulateurs de la transcription des gènes. Cependant, peu d’études ont été menées sur la dynamique de ces complexes au niveau cellulaire et sur les mécanismes régulant leur assemblage. Au cours de mes travaux de thèse, j’ai utilisé des approches d’imagerie sur cellules vivantes, afin de déterminer la mobilité de ces complexes en comparaison avec celle d’autres régulateurs transcriptionnels. Les résultats ont montré que les sous-unités de SAGA et ATAC interagissent de manière transiente avec la chromatine. En complément, nous avons montré que les sous-unités spécifiques de SAGA et ATAC (ADA2b et ADA2a) ont des propriétés dynamique intracellulaire différentes et que GCN5, affecte la distribution d’ADA2a. Des analyses protéomique menées sur le comportement de ces protéines au niveau endogène, ont permis de montrer que les voies d’assemblage de ces deux complexes étaient différentes au niveau cytoplasmique et nucléaire. / Human SAGA and ATAC, are histone acetyltransferase (HAT) containing complexes that share a set of subunits and facilitate RNA polymerase II (Pol II) transcription. Little is known for the dynamics of the complexes in living cells and the regulation of their assembly. In this work, we used live-cell imaging to characterise the mobility of the two complexes and compare it with other actors of Pol II transcription. All tested ATAC and SAGA subunits exhibit very transient interactions with chromatin, a property that explains certain aspects of the function of the complexes. Moreover, we showed that overexpressed ATAC- and SAGA-specific HAT-module subunits (ADA2a and ADA2b respectively) have different intracellular dynamics and that the abundance of the shared subunit GCN5, affects the distribution of ADA2a. Quantitative proteomic analysis expanded our findings on endogenous proteins and provided evidence that the cytoplasmic and nuclear assembly pathways of SAGA and ATAC are different.

Reactivation of latent infection is the major cause of tuberculosis (TB). Cholesterol is a critical carbon source during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the sterol-ring degradation and is essential for intracellular survival. NAT from M. tuberculosis (TBNAT) can utilise propionyl-CoA and therefore was proposed as a target for TB-drug development. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. NAT inhibitors, including the piperidinol class, were identified by high-throughput screening. The insolubility of recombinant TBNAT has been a major limitation in pursuing it as a drug target. Subcloning tbnat into a pVLT31 vector resulted in a yield of 6-16 mg/litre-bacterial-culture of pure-soluble recombinant TBNAT. The increased yield allowed for extensive screening for crystallisation conditions. However, since a structure was not obtained, the model NAT from M. marinum (MMNAT) was employed to further understand NAT as a target. Screening against a panel of Acyl-CoA cofactors showed that MMNAT can also utilise propionyl-CoA. The MMNAT structure in complex with the high affinity substrate hydralazine was determined (2.1 Å) and the architecture of the arylamine pocket was delineated. A novel mechanism for the acetylation reaction of hydralazine has emerged. It is proposed that the acetyl group is transferred from acetyl-CoA to the heterocyclic aromatic nitrogen of hydralazine, which explains the immediate cyclisation of the acetylated metabolite into an N-methyltriazolophthalazine. By employing mass spectroscopy, enzyme assays, computational docking and structural studies, a covalent mechanism of inhibition by the piperidinol class was established, and the inhibitor-binding pocket was identified. Inhibitors with new scaffolds were identified using the in silico 3D-shape screening and thermal shift assay.

Thesis (PhD (Molecular Biology and Human Genetics))--University of Stellenbosch, 2005. / 157 leaves single sided printed, preliminary pages i-xvii and numbered pages 1-141. Includes bibliography, and abbreviations and a list of figures. / ENGLISH ABSTRACT: A gene coding for Arylaminie N-acetyltransferase (NAT) has been found in Mycobacterium tuberculosis, the casual agent of tuberculosis (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 of the 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 of nat 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 of the 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. 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 of mycobacteria. 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 of NAT could result in hindered growth and hence spread of the 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.

Flavonoids are natural compounds commonly ingested in herbs and vegetables. They are believed to have a positive impact on human organism, in particular by their antioxidant, hepatoprotective and anti-cancer effects. In these days, it is possible to consume high concentrations of these compounds in form of dietary supplements. However it is not clear, whether flavonoids in such unnaturally high concentrations are still beneficial or rather harmful. It has already been proven, that flavonoids can influence the activity of biotransformation enzymes and interfere e.g. with the process of carcinogenesis and drug metabolism. For that reason it is important to investigate the impact of an increased intake of flavonoids. The aim of this thesis was to investigate the influence of dihydromyricetin (a potential drug to cure alcohol use disorder) and its structurally similar flavonoid myricetin on the activity of enzymes, cytochrome P450 2E1 (CYP2E1) and N­acetyltransferases 1 and 2 (NAT1/2). The research included the determination of the impact of a premedication by these flavonoids on the expression and activity of CYP2E1 and NAT1/2. The inhibition capacity of myricetin and dihydromyricetin towards the activity of CYP2E1 and NAT1/2 was also investigated. After the flavonoid premedication of rats the...