The plant phenylpropanoid pathway is initiated from deamination of phenylalanine to form cinnamic acid followed by hydroxylation and methylation of the aromatic ring to generate a variety of phenolic compounds including lignin monomers, flavonoid compounds and sinapate esters. The incorporation of phenylpropanoid metabolism served as a key step in the early land-colonization of plants from aqueous environment since phenolic compounds play important roles in plant development and abiotic/biotic stress responses. Lignin is a heteropolymer of hydroxycinnamyl alcohols that are derived from the major branch of plant phenylpropanoid pathway. The main function of lignin is to enhance the strength of plant cell wall and waterproof the vascular system for long-distance transportation of water and solutes. In addition, lignin is also involved in protecting plants against pathogen attack. My Ph.D. research is to investigate how lignin biosynthesis contributes to plant immunity. The results showed that the expression of major lignin biosynthetic genes was induced upon host fungal pathogen infection. Moreover, a mutant disrupted in the lignin gene F5H1 showed enhanced susceptibility when challenged with several fungal pathogens. F5H1 encodes a ferulic acid 5-hydroxylase that is uniquely present in angiosperm plants, leading to the biosynthesis of syringyl lignin monomer, which is not present in gymnosperm plants. Subsequent research demonstrated that f5h1 mutation impaired the penetration (pre-invasion) resistance but did not impact post-invasion resistance. Furthermore, the pathogen-induced expression of lignin genes was independent of well-characterized defensive signaling pathways, and regulated by a novel regulating mechanism. F5H1 contributes to pmr2-mediated resistance but acts independently of other molecular components of penetration resistance including PEN1, PEN2, and PEN3. In contrast to f5h1, a knockout mutant of flavonoid pathway gene chalcone isomerase (CHI/TT5) showed enhanced resistance to host anthracnose pathogen Colletotrichum higginsianum in a salicylic acid (SA)-dependent manner. Taken together, our results for the first time provide genetic evidence demonstrating that lignin biosynthetic gene F5H1 plays critical roles in plant penetration resistance and that an uncharted pathway in flavonoid metabolism confers an SA-dependent resistance pathway in Arabidopsis.

The dissertation thesis deals with the problems of the use of plasma technologies in agriculture for the purpose of improving the quality of feedstuffs for agricultural animals. The attention is focused above all on the use of low-temperature gliding arc plasma discharge generated by the atmospheric pressure for the elimination of model fungal pathogens (T. virens) and for the testing of the possibility of the use of plasma for the decreasing of mycotoxin load (DON and D3G). The professional orientation of the dissertation thesis is strongly interdisciplinary with the overlap to the area of the physics of plasma, microbiology, zootechnics and the proposal and construction of technical facilities. With that corresponds also the division of thesis into individual chapters. In the research part, there is a summary of actual pieces of knowledge from the area of fungal microorganisms and mycotoxins produced by these with the view on their presence in feedstuffs. There follows a research of pieces of knowledge of low-temperature plasma and its applications in the food industry and feeding. In the chapter Material and Methodology, the applicated methods of work, employed instruments and biological material are described. The resulting part is, for its clear arrangement, divided into five subchapters ? stating of selected parameters of plasma device, influence of plasma on nutritional parameters, influence of plasma on the model fungus Trichoderma virens and possibilities of the use of plasma discharge for the decreasing of concentration of mycotoxins in feedstuffs. The final part is dedicated to the description of system which enables the treatment of samples in continuous regime. The treatment of mycelial discs by plasma discharge lead to the decrease of germination capacity of spores of fungi, while there was experimentally proven the synergic effect of the influence of individual mechanisms by which the plasma interacts with the surface of biological material. At the same time, there was not observed a more significant change in the composition of feedstuffs, more significant is only the change of humidity of sample. In the laboratory conditions, the decreasing of concentration of mycotoxins spread on laboratory glass was proven. At the samples of feedstuffs contaminated by natural way, it was not successful to reliably provedecreasing of concentration of mycotoxins in a sample as a result of plasma activity.

<p>Sexual reproduction in fungi is governed by a specialized genomic region called the mating-type locus (MAT). The ascomycetes, the largest phylum of fungi, primarily possess a bipolar mating system while the basidiomycetes, the second largest group, are mostly tetrapolar. The human fungal pathogen and basidiomycetous yeast Cryptococcus neoformans has evolved a bipolar mating system that encodes homeodomain (HD) and pheromone/receptor (P/R) genes. The MAT locus of C. neoformans is unusually large, spans greater than 100 kb, and encodes more than 20 genes. To understand how the pathogenic Cryptococcus species complex evolved this unique bipolar mating system, we investigated the evolution of MAT in closely and distantly related species and discovered an extant sexual cycle in Cryptococcus amylolentus. </p><p>Phylogenetic analysis using a six-gene multi-locus sequencing (MLS) approach identified the most closely related species to the pathogenic Cryptococcus species complex that are currently known. The two non-pathogenic sibling species, Tsuchiyaea wingfieldii and Cryptococcus amylolentus, and the more distantly related species Filobasidiella depauperata define the Filobasidiella clade. We also resolved the phylogeny of the species located in the sister clade, Kwoniella. A comprehensive tree dendrogram revealed that the 15 Tremellales species examined suggests a common saprobic ancestor. Moreover, the pathogenic Cryptococcus species have a saprobic origin but later emerged as pathogens. We further characterized the mating-type locus for T. wingfieldii and C. amylolentus by cloning and sequencing two unlinked genomic loci encoding the HD and P/R genes. Interestingly, linked and likely divergently transcribed homologs for SXI1 and SXI2 are present in T. wingfieldii and C. amylolentus, while the P/R alleles contain many genes also found in the MAT locus of the pathogenic Cryptococcus species. Also, hypothetical genes present in C. neoformans MAT are also MAT-linked in both species and indicate a possible translocation event between chromosomes 4 and 5 of C. neoformans. Our analysis of MAT in the sibling species indicates that T. wingfieldii is likely tetrapolar, and the C. amylolentus sequence comparison of the dimorphic SXI1 and SXI2 region and the pheromone receptor, STE3, suggests that C. amylolentus is also tetrapolar. The examination of MAT in these sibling species confirms the model for MAT evolution previously proposed in which this structure in C. neoformans and C. gattii evolved from an ancestral tetrapolar mating system. Moreover, the organization of MAT in these sibling species mirrors key aspects of the proposed intermediates in the evolution of MAT in the pathogenic Cryptococcus species, and for sex chromosomes in plants, animals, and alga in general. </p><p>We discovered an extant sexual cycle for C. amylolentus, a species previously thought to be asexual. Matings between two strains of opposite mating-types produce dikaryotic hyphae with fused clamp connections and uni- and bi-nucleate basidiospores. Genotyping of basidiospores using markers linked and unlinked to MAT revealed that genetic exchange (recombination) occurs during the sexual cycle of C. amylolentus, and it is likely that either aneuploids are generated during sex or more than one meiosis event occurs within each basidium. This is in contrast to C. neoformans, where only one meiotic event per basidium has been observed. Uniparental mitochondrial inheritance has also been observed in C. amylolentus progeny; similar to the pathogenic Cryptococcus species, mtDNA is inherited from the C. amylolentus MATa parent. Analysis of sex in C. amylolentus has provided insight into the mechanisms that phylogenetically related fungi employ in orchestrating sexual reproduction. </p><p>We also extended our analysis to include the distantly related tetrapolar basidiomycete Tremella mesenterica. We completed comparisons of MAT-specific genes between five strains of T. mesenterica and identified the regions that define its mating-type system. The HD locus is limited to the SXI1- and SXI2-like genes while the P/R locus is defined by STE3, STE12, STE20, and the pheromone gene, tremerogen a-13. Interestingly, many of the genes associated with the MAT locus of the pathogenic Cryptococcus species flank the HD and P/R locus and are not incorporated in MAT in T. mesenterica. The MAT region includes transposons and C. neoformans hypothetical genes also present in T. wingfieldii and C. amylolentus. The mating-type system in T. mesenterica reflects an ancestral intermediate in the evolution of the MAT locus in the pathogenic Cryptococcus species. In conclusion, this study provides an in-depth analysis on the structure, function, and evolution of an unusual mating-type locus with broader implications for the transitions in modes of sexual reproduction in fungi that impact gene flow in populations.</p> / Dissertation

Considerable changes have occurred in recent years, regarding the taxonomy and ecology of Eucalyptus fungal pathogens previously treated in the genera Cryphonectria and Endothia. Cryphonectria cubensis now resides in Chrysoporthe with two species, which are very distinct from Cryphonectria. The fungus previously known as E. gyrosa was moved to C. eucalypti and will soon be known as Holocryphia eucalypti. It is very likely that C. eucalypti and Chr. cubensis were introduced onto the African continent, but the hypothesis remains to be tested, while Chr. austroafricana seems native to the African continent. The aim of studies contained in this thesis was to consider the distribution, taxonomy and diversity of Chrysoporthe spp. and Cryphonectria eucalypti on the African continent. This was achieved through surveys in southern and eastern Africa, of both Eucalyptus spp. and native tree species belonging to the Myrtales. The intention was that the results of the studies in this thesis should aid in a better understanding of the taxonomy, origin, distribution, host range, as well as pathogenicity of various Cryphonectria and Chrysoporthe species in eastern and southern Africa. Various new hosts, new areas of occurrence and taxonomic changes have occurred for species of Cryphonectria sensu lato, previously known only on Eucalyptus spp. Chapter one of this thesis presented an overview of the most recent findings regarding the taxonomy, host range and distribution of C. cubensis sensu lato and C. eucalypti. The background to the description of a new genus, Chrysoporthe Gryzenhout&M.J. Wingf. and three new species namely; Chr. cubensis, Chr. austroafricana and Chrysopothella hodgesiana, previously considered to represent C. cubensis was also considered. Furthermore, the wide host range of Chrysoporthe spp. has been reviewed. The fungi are known on various genera in the order Myrtales in both tropical and subtropical areas, worldwide. Emphasis was placed on these Eucalyptus pathogens in Africa. Chrysoporthe cubensis and Chr. austroafricana, collectively known as Cryphonectria cubensis in the past, are important canker pathogens of Eucalyptus spp. worldwide. In chapter two of this thesis I have shown, for the first time, that Chr. cubensis occurs in Kenya, Malawi and Mozambique on non-native Eucalyptus spp. and Chr. austroafricana occurs in Mozambique, Malawi and Zambia on non-native Eucalyptus spp. and native S. cordatum. I was also able to show that Chr. austroafricana causes cankers at the base and higher up on stems of Eucalyptus trees in South Africa and Malawi, which is contrary to prior knowledge. Likewise, the sexual state of this fungus has been shown to be equally abundant as the asexual state in countries north of South Africa, contrary to the situation in southern Africa where the asexual state predominates. The known distribution range of Chr. austroafricana within South Africa was also expanded through this study. Chrysoporthe cubensis is an important fungal pathogen of Eucalyptus spp., worldwide. The fungus is also known on many other hosts all residing in the order Myrtales. Previous surveys conducted in eastern and southern Africa to assess the distribution of Chrysoporthe spp. in this region, revealed the occurrence of Chr. cubensis on Eucalyptus spp. in Kenya, Malawi and Mozambique. In chapter three of this thesis, the population structure of Chr. cubensis isolates from Eucalyptus spp. from Kenya, Malawi and Mozambique was considered for the first time. This represents a first attempt to consider the genetic structure of the fungus from eastern Africa. Results show that there is a very low genetic diversity within the populations of Chr. cubensis from Kenya, Malawi and Mozambique, implying that the fungus is probably newly introduced in these areas. Based on phylogenetic analyses, the origin of eastern African Chr. cubensis is most likely Asia. In chapter four of this thesis, polymorphic microsatellite DNA markers were developed from a single spore isolate of C. eucalypti collected from Eucalyptus stem canker in South Africa. Markers were obtained using the enrichment technique known as FIASCO (Fast Isolation by AFLPs of Sequences Containing Repeats). Ten polymorphic markers were isolated, of which 2 were discarded due to their high polymorphism in the flanking region. These markers will consequently provide useful tools for future investigations considering the population biology and especially the global spread of C. eucalypti. Cryphonectria eucalypti is a fungal pathogen considered opportunistic in South Africa, while in Australia it has been associated with sporadic but serious disease problems. Chapter five of this thesis presents results on the population structure of C. eucalypti from South Africa, eastern and western Australia. Nei's gene diversity (H) showed that the eastern Australian population is most genetically diverse and the western Australian populations from Corymbia and Eucalyptus somewhat less diverse. The South African population displayed the lowest genetic diversity. The high genetic diversity in the Australian populations supports the view that C. eucalypti is native to that region. This is consistent with the fact that Eucalyptus species are also native to the Australian continent. In chapter six of this thesis, I have shown that the fungus isolated from H. canescens, S. cordatum and T. granulosa in South Africa represents a new genus and species related to, but distinctly different from Chrysoporthe. Celoporthe dispersa gen. et sp. nov. is, therefore, described to accommodate this fungus. This description was supported by both morphological characteristics and DNA sequence data. These have clearly shown that isolates of C. dispersa form a clade distinct from Chrysoporthe, Holocryphia and other taxa, which it resembles morphologically. Pathogenicity tests showed that C. dispersa is not pathogenic to H. natalensis, but a potential pathogen of Eucalyptus and Tibouchina spp. The collection of studies included in this thesis demonstrated that Chrysoporthe spp. occur in Malawi, Mozambique, Zambia, Kenya and Tanzania on both Eucalyptus and native Syzygium cordatum trees. This significantly expands the geographical distribution of these important pathogens. The studies have also shown that Chrysoporthe cubensis has recently been introduced on the continent. It is my hope that new knowledge emerging from studies in this thesis will aid in quarantine measure to control the spread of these important fungal pathogens including the new species Celoporthe dispersa. / Thesis (PhD)--University of Pretoria, 2008. / Microbiology and Plant Pathology / PhD / Unrestricted

>Magister Scientiae - MSc / The high rate of rediscovery of known compounds has led to a decline in the discovery of novel natural products. The high biodiversity of organisms growing in extreme conditions such as oceans has led to the increased interest by researchers for their use as a source of novel natural products. Marine bacteria are known for their extensive biosynthetic capacity to produce diverse natural products, which are suitable for various biotechnology applications such as in agriculture, for treatment of fungal plant pathogens, and as antibiotics, for treatment of bacterial infections. This study aimed at discovering novel secondary metabolites from marine bacteria previously associated with novel marine invertebrate species endemic to the South African coast. The methodologies used in this study included a bioassay guided fractionation coupled to genome sequencing and mining. For the bioassay guided fractionation approach, the study first focused on screening marine bacteria for antimicrobial activity when cultured on 4 different media, against fungal strains previously shown to be virulent olive trunk pathogens. In parallel, the bacterial isolates with the most inhibitory activity against the fungal pathogens were also screened for antimicrobial activity against 4 indicator strains including Gram-negative Escherichia coli 1699 (E. coli), Pseudomonas putida, and Gram-positive Staphylococcus epidermidis ATCC14990, and Bacillus cereus ATCC10702. One of the marine bacterial isolates, PE6-126, showed diverse antimicrobial activity including antibacterial and antifungal activity against the tested strains. The genome sequencing data revealed that this isolate was B. cereus based on the average nucleotide identity (ANI) (>99%) to reference strains. antiSMASH analysis of the genome revealed nine predicted secondary metabolite clusters including bacteriocins (2), non-ribosomal peptide synthetase (NRPS) (2), siderophore (1), sactipeptide (1), betalactone (1), linear azol(in)e-containing peptides (LAP) - bacteriocin (1) and a terpene (1). Some of these pathways had low to no sequence similarity to known pathways, indicating the potential of these pathways to produce novel compounds. One of the pathways showed very high sequence similarity to the thuricin CD pathway in Bacillus thuringiensis. Considering that thuricin CD has been reported to have antimicrobial activity against B. cereus (ATCC1072), it was hypothesised that it could also be produced by PE6-126. However, the antimicrobial extract from PE6-126 was tested for sensitivity to proteinase K and heat treatment, which thuricin CD is known to be sensitive to. The results revealed that the antimicrobial activity was not lost after treatment, implying that a different metabolite could be responsible for the anti-B. cereusactivity. In addition, PE6-126 initially displayed antimicrobial activity against a multi-drug resistant E. coli 1699, suggesting some of the antimicrobial compound/(s) produced by this strain could potentially be novel. The bioassay-guided fractionation approach coupled to Liquid Chromatography Mass Spectrometry (LC-MS) did not lead to identification of the antimicrobial compound/(s), therefore it remains a question whether the secondary metabolite pathways predicted by antiSMASH lead to the production of the active compound/(s).The results from this study showed that even well studied species have the potential to synthesize as yet undescribed compounds, based on the novelty of some of the pathways. This study highlights the importance of employing a genome-guided approach in drug discovery, as there may be many novel compounds to discover from biosynthetic pathways that have not yet been characterised. Further research is needed to identify the antimicrobial compound/(s) produced by PE6-126.

Diseases caused by bacteria, fungi and viruses pose a significant threat especially to poor rural communities. Viral infections are frequently complicated by secondary bacterial and fungal infections which remain a major challenge globally and in particular, in sub Sahara Africa amongst humans and animals alike. The main aim of this study was to develop a low toxicity plant extract or isolated compound active against viral, bacteria and fungal pathogens from selected plant species. Seven tree species that were investigated were Acokanthera schimperi, Carissa edulis, Ekebergia capensis, Podocarpus henkellii, Plumbago zeylanica, Annona senegalensis and Schrebera alata traditionally used in the treatments of various ailments were selected and extracted using solvents of varying polarity. Extracts of selected plants were tested for activity against two Gram positive and two Gram negative bacterial namely Enterococcus faecalis and Staphylococcus aureus and two Gram-negative species, Pseudomonas aeruginosa and Escherichia coli respectively, three fungal pathogens: Candida albicans, Cryptococcus neoformans and Aspergillus fumigates and four enveloped animal viruses: feline herpes virus–1 (FHV-1, dsDNA), canine distemper virus (CDV, ssRNA), canine parainfluenza virus-2 (CPIV-2, ssRNA) and lumpy skin disease virus strain V248/93 (LSDV, dsDNA). The presence of antioxidant constituents in the different extracts and cytotoxicity against three cell types CRFK, bovine dermis and Vero cells were determined. Bioautography and the serial microplate dilution methods were used to determine the number of antimicrobial compounds and antimicrobial activity of extracts against bacterial and fungal pathogens. Virucidal and attachments assays were used to determine the activity against viral pathogens. Qualitative antioxidant activities of extracts were tested using the DPPH reagent and cytotoxicity using the MTT assay. Biological activity was observed in all the extracts against one or more organisms on bioautography. The intermediately polar system (CEF) separated more active constituents. Some extracts had compounds with similar Rf values active against one or more organisms. In both the antibacterial and antifungal assays, acetone extracts had the highest activity followed by DCM against one or more pathogens. Hexanes extracts were the least active. P. henkellii extracts had more active compounds against the bacteria and Annona senegalensisagainst the fungi. In the micro-dilution assay, S. aureus was the most susceptible bacterial organism to extracts of the different plant, followed by P. aeruginosa andEscherichia coli, and E. faecalis the least. C. neoformans on the other hand was the most susceptible fungal pathogen. In the antiviral assay, although activity was observed with hexane extracts of some plants in the virucidal assay, the most potent inhibition was observed with the acetone and methanol extracts of Podocarpus henkelii against CDV and LSDV in the virucidal assay and acetone extracts in the attachment assay. In general the hexane was the least toxic while the intermediate polarity extracts were generally the most toxic indicating that highly polar compounds were possibly poorly or highly absorbed through membranes in the former and later respectively. Of the three cell types used CRFK was the most sensitive followed by bovine dermis and Vero cells the least. Cytotoxicity studies of extracts of the different plants revealed A. senegalensis and A. schimperi extracts were the most toxic plants in the cellular assay. These plants are toxic to animals and the cytoxicity is in line with the in vivo toxicity. The protective effects of antioxidant constituents in some extracts varied and appear to be influenced by the metabolism of the type of cell in culture. It also appears to suggest that metabolism in kidney derived cells can be influenced by species variation in the origin of cells. P. henkellii was selected for isolation of bioactive compound. Three compounds were isolated and their structure elucidated using 13C and 1H NMR and mass spectrometric data. The antibacterial, antifungal and antiviral activity of the isolated compounds 7’, 4’, 7’’, 4’’’, tetramethoxy amentoflavone (C1), isoginkgetin (C2) and Podocarpusflavone–A (C3) were determined. Compound C2 was the most active against E. coli and S. aureus (MIC = 60 ìg/mE) and a selectivity index (SI) value of 16.67. The compound was also active against A. fumigatus and C. neoformans (SI = 33.33) suggesting both antibacterial and antifungal activity with relative safety. Compound C3 had a broad spectrum of activity against E. faecalis and P. aeruginosa with SI values of 4. A less potent activity of the compounds was obtained in both the virucidal and attachment assays against test pathogens, indicating the lower activity of the compounds against tested viral pathogens. The studies further suggest structural activity relationship in the antimicrobial activity of biflavonoids. The compounds C1 and C2 had no toxic effect on the three cell types and mutagenicity studies indicated no activity of these compounds. Podocarpusflavone-A occurs in every species of Podocarpus so far investigated, except P. latifolius. These studies represent the first isolation of bioactive compounds from P. henkellii. Although a different extractant was used than that used by traditional healers, the presence of antiviral compounds in Podocarpus henkelii against two unrelated viruses may justify on a chemotaxonomic basis the traditional use of related species Podocarpus latifoliusand Podocarpus falcatus in the traditional treatment of canine distemper infection in dogs. / Thesis (PhD)--University of Pretoria, 2011. / Paraclinical Sciences / unrestricted

The health of pollinators, especially bees, is of the utmost importance to success of many agricultural ecosystems. Microorganisms can cause diseases in bees; such microbes are pathogenic. The ability of a pathogen to cause harm to its host (such as a bee) is termed its virulence. Studying the evolution of different levels of virulence can lead researchers to a better understanding of pathogens, and potentially predict how much harm a pathogen can cause in the future. We studied the evolution of virulence levels for a fungal disease of bees. This group of fungi is composed of 28 species, and some cause a disease in bees called chalkbrood while others do not. Using what we know about virulence evolution we wanted to see if the pathogens could infect all bees, if the pathogens varied in virulence when infecting at the same time as another pathogen, and if solitary bees had any behavioral adaptations that might increase or decrease chalkbrood infection. By using DNA sequences, the relationship between the genetic structures of each of the fungal species was studied, and we found that pathogens of solitary bees grouped together while pathogens of social bees (honey bees) were not part of this group. We then found that a solitary bee pathogen did not infect honey bees very well, and vice versa. The nuances of the relationship between two solitary bee pathogens were examined more closely to determine how the two pathogens interact in this bee. In this case, under varying conditions of infection, one pathogen always maintained a similar level of virulence and spore production, while the other pathogen varied in these measures. In addition, when doses of these fungi were fed to bee larvae at different times, more bees survived than when the doses were given at the same time, suggesting that bee immune responses are very important. Finally, we found no evidence of any specific behaviors of solitary bees exposed to infective spores that would suggest these bees have behaviors that are evolved to alter chalkbrood levels in populations.

Among fungal pathogens responsible for opportunistic infections, species of the genus Candida have a major role (Mitchell, 1998). Various Candida species cause superficial infections which can be cured by the currently available antifungal arsenal (Noble and Johnson, 2007). However, species of the genus Candida are also responsible for life-threatening systemic infections, particularly in immunocompromised patients with weakened immune system. Among Candida species, C. albicans, which can also be a commensal of the skin and the gastrointestinal and genitourinary tracts, is responsible for the majority of Candida bloodstream infections. However, there is an increasing incidence of infections caused by C. glabrata because it is less susceptible to azoles. Other medically important Candida species include C. parapsilosis, C. tropicalis and C. dubliniensis. The problem has been further worsened by the emergence of many drug resistant isolates which pose a major hurdle during a given treatment regimen. Therefore, there is a dire need to identify novel drug targets and the current study focuses on one such protein found in C. albicans and related Candida species. CaGAL102 does not encode a functional galactose epimerase CaGAL102 was previously identified in the lab as a paralog of CaGAL10. CaGAL10 endoes a functional UDP-galactose 4-epimerase and it can complement a Scgal10 null strain. Further, work on the Gal10 protein in the encapsulated yeast Cryptococcus neoformans identified two Gal10 paralogs in the genome, Uge1 and Uge2 with distinct functions (Moyrand et al., 2008). A similar scenario is found in S. pombe in which two Gal10 sequence homologs have been annotated. In the light of these observations, we wanted to test if CaGAL102 also encodes a functional ScGAL10 homolog. We found that CaGAL102 could not complement Scgal10 null strain though there was a strong conservation in the cofactor and the catalytic motif in both the proteins. We found after a careful literature review that Gal10 belongs to a family of proteins called the short chain dehydratase/reductase family (SDR) (Jornvall et al., 1995), members of which are characterised by the presence of glycine rich cofactor binding motif at the N-terminus and an YXXXK catalytic motif. Proteins belonging to the SDR family have a residue level identity of 15-30% indicating early duplication and divergence. Based on our literature survey we carried out a BLAST search in the NCBI protein database using CaGal102 as the bait protein. We found that CaGal102 is 32% identical at the protein level to dTDP-glucose 4,6 dehydratase (RmlB), another member of the SDR family. RmlB is the second enzyme of the rhamnose biosynthetic pathway which gives rise to dTDP-rhamnose. This pathway is involved in cell wall biosynthesis in bacteria and it has been shown that rmlB is essential for growth of Mycobacterium smegmatis (Li et al, 2006). Interestingly rhamnose is not present in the cell wall of C. albicans. Biochemical characterisation of CaCaGal102 A plant homolog of RmlB is found in A. thaliana which uses UDP-glucose as the substrate (Oka et al., 2007). Based on our alignment data we identified many critical residues in CaGal102. Most importantly we identified that lysine at position 159 lies in the YXXXK motif and could be important for activity. We therefore, mutated the lysine at position 159 to alanine. In order to find out the biochemical function of CaGal102 in vitro, we cloned expressed and purified recombinant wild type and catalytic mutant proteins from E. coli and used the purified proteins for our assays. We found that CaGal102 uses UDP-glucose as the preferred substrate. To further substantiate our data, we reintegrated the wild type or the mutant alleles in the native locus of CaGAL102 and checked for the rescue of morphology defects like filamentation and sensitivity to cell wall damaging agents. We also found that the Cagal102∆/∆ strain is avirulent in a mouse model of systemic infection. We have also carried out infection studies with the null mutant and the wild type and the catalytic mutant reintegrant strains. Our observation suggests that reintegrating one copy of the wild type allele rescues the virulence defect. Interestingly the strain harbouring one copy of the mutant allele behaves like the null mutant in a mouse model of systemic infection. We have also identified sequence homologs of CaGal102 in related Candida species. It is plausible to think that the homologs in related species also have similar effects and hence targeting this protein by a small molecule could help in treating candidiasis caused by related species. CaGAL102 is involved in cell wall architecture in C. albicans To elucidate the role of CaGal102 in C. albicans we generated a knockout out strain and studied various mutant phenotypes. The most striking observation was that the cells of the null mutant were filamentous as compared to the wild type control when grown in normal rich media. Further the cells were sensitive to various cell wall damaging agents and also to hygromycin B. We reasoned that lack of CaGal102 causes perturbation in the cell wall architecture rendering the cells sensitive to various cell wall damaging agents. To further strengthen this hypothesis, we decided to study the genetic interaction of CaGAL102 with genes known to be involved in cell wall biosynthesis in C. albicans. One of the candidate genes we chose for our study was GAL10, deletion of which in C. albicans renders the cells sensitive to various cell wall damaging agents. Loss of function of UGE1 in C. neoformans impaired biosynthesis of a cell wall component, galactoxylomannan. We found that cells lacking both Gal102 and Gal10 adhered to nylon membranes poorly as compared to single mutants or the wild type control. The second gene we chose was a P-type ATPase, PMR1 deletion of which causes increased sensitivity to cell wall damaging agents and hyper-activation of the cell wall integrity pathway similar to Cagal102∆/∆ strain. We found that cells lacking both Pmr1 and Gal102 were more sensitive to hygromycin B as compared to the single mutants. This confirmed our idea that CaGal102 is a novel gene involved in cell wall biogenesis in C. albicans. REFERENCES: Mitchell, A.P. (1998) Dimorphism and virulence in Candida albicans. Curr Opin Microbiol, 1, 687-692. Noble, S.M. and Johnson, A.D. (2007) Genetics of Candida albicans, a diploid human fungal pathogen. Annu Rev Genet, 41, 193-211. Moyrand, F., Lafontaine, I., Fontaine, T. and Janbon, G. (2008) UGE1 and UGE2 regulate the UDP-glucose/UDP-galactose equilibrium in Cryptococcus neoformans. Eukaryot Cell, 7: 2069-2077. Jornvall Hans, Persson Bengt, Krook Maria,‟ Atrian Silvia, Gonzalez-Duarte Roser, Jeffery Jonathan, and Ghosh Debashis (1995). Short-Chain Dehydrogenases Reductases (SDR). Biochemistry, 34: 6004-13. Li, W., Xin, Y., McNeil, M.R. and Ma, Y. (2006) rmlB and rmlC genes are essential for growth of mycobacteria. Biochem Biophys Res Commun, 342: 170-178. Oka, T., Nemoto, T. and Jigami, Y. (2007) Functional analysis of Arabidopsis thaliana RHM2/MUM4, a multidomain protein involved in UDP-D-glucose to UDP-L-rhamnose conversion. J Biol Chem, 282: 5389-5403.

>Magister Scientiae - MSc / The high rate of rediscovery of known compounds has led to a decline in the discovery of novel natural products. The high biodiversity of organisms growing in extreme conditions such as oceans has led to the increased interest by researchers for their use as a source of novel natural products. Marine bacteria are known for their extensive biosynthetic capacity to produce diverse natural products, which are suitable for various biotechnology applications such as in agriculture, for treatment of fungal plant pathogens, and as antibiotics, for treatment of bacterial infections. This study aimed at discovering novel secondary metabolites from marine bacteria previously associated with novel marine invertebrate species endemic to the South African coast. The methodologies used in this study included a bioassay guided fractionation coupled to genome sequencing and mining. For the bioassay guided fractionation approach, the study first focused on screening marine bacteria for antimicrobial activity when cultured on 4 different media, against fungal strains previously shown to be virulent olive trunk pathogens. In parallel, the bacterial isolates with the most inhibitory activity against the fungal pathogens were also screened for antimicrobial activity against 4 indicator strains including Gram-negative Escherichia coli 1699 (E. coli), Pseudomonas putida, and Gram-positive Staphylococcus epidermidis ATCC14990, and Bacillus cereus ATCC10702. One of the marine bacterial isolates, PE6-126, showed diverse antimicrobial activity including antibacterial and antifungal activity against the tested strains. The genome sequencing data revealed that this isolate was B. cereus based on the average nucleotide identity (ANI) (>99%) to reference strains. antiSMASH analysis of the genome revealed nine predicted secondary metabolite clusters including bacteriocins (2), non-ribosomal peptide synthetase (NRPS) (2), siderophore (1), sactipeptide (1), betalactone (1), linear azol(in)e-containing peptides (LAP) - bacteriocin (1) and a terpene (1). Some of these pathways had low to no sequence similarity to known pathways, indicating the potential of these pathways to produce novel compounds. One of the pathways showed very high sequence similarity to the thuricin CD pathway in Bacillus thuringiensis. Considering that thuricin CD has been reported to have antimicrobial activity against B. cereus (ATCC1072), it was hypothesised that it could also be produced by PE6-126. However, the antimicrobial extract from PE6-126 was tested for sensitivity to proteinase K and heat treatment, which thuricin CD is known to be sensitive to. The results revealed that the antimicrobial activity was not lost after treatment, implying that a different metabolite could be responsible for the anti-B. cereus activity. In addition, PE6-126 initially displayed antimicrobial activity against a multi-drug resistant E. coli 1699, suggesting some of the antimicrobial compound/(s) produced by this strain could potentially be novel. The bioassay-guided fractionation approach coupled to Liquid Chromatography Mass Spectrometry (LC-MS) did not lead to identification of the antimicrobial compound/(s), therefore it remains a question whether the secondary metabolite pathways predicted by antiSMASH lead to the production of the active compound/(s). The results from this study showed that even well studied species have the potential to synthesize as yet undescribed compounds, based on the novelty of some of the pathways. This study highlights the importance of employing a genome-guided approach in drug discovery, as there may be many novel compounds to discover from biosynthetic pathways that have not yet been characterised. Further research is needed to identify the antimicrobial compound/(s) produced by PE6-126.

Infectious diseases are the world’s leading cause of premature deaths in humans and animals. The resistance to antibiotics and the emergence of new infectious diseases has increased the need for additional effective antimicrobial products. Despite numerous publications investigating antimicrobial activity of plant extracts it appears that no effective single product antimicrobial has yet been developed from plants. In many cases, however crude plant extracts have excellent activity and may provide useful products. Plants are frequently selected based on traditional use. Traditional healers usually use aqueous extracts of plants which in our experience generally have very low activities and it may be one of the reasons why no new products were developed from plants. Another approach to select plants for research is to use the taxonomic approach based on the premises that: (1) there is a correlation between active chemical compounds and antimicrobial activity; and (2) species in a family or order may have similar activities if the chemical precursors are inherited from a common ancestor. Future screening programmes could then concentrate on close relatives of species within these promising families and orders. The main aim of this study was to randomly screen leaf extracts of several hundred southern African tree species against important microbial pathogens to determine which taxa have the highest activity and may yield useful products to treat infections in human and animal health markets. A wide selection of plant species improved the possibility of finding promising extracts and has the advantage that active compounds may be discovered from plants that are not used traditionally. To ensure sustainable use only leaves of trees were examined. A spin off of this study would also indicate the susceptibility of different organisms, correlate the antimicrobial activities of the different organisms and determine what minimum inhibitory concentration (MIC) represents a good activity based on investigating many extracts against many microbes. The antimicrobial activity was determined by using a sensitive serial dilution microplate method. Acetone extracts were tested against two Gram-positive bacteria, two Gram-negative bacteria and two fungi, i.e. Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Cryptococcus neoformans. Small and mostly insignificant differences were found between the susceptibility of the microbial pathogens to the extracts. E. faecalis was the most sensitive bacterium and C. neoformans the most sensitive fungal organism. The strongest correlations in activities among the pathogens were between C. albicans and C. neoformans, and among the pathogen classes between Gram-positive and Gram-negative bacteria. The tree extracts analysed in the present study had a wide range of activities against the different pathogens. Twenty six per cent of the extracts inhibited the pathogens at MIC levels of 0.16 mg/ml. This clearly shows that 0.16 mg/ml is not low enough to discriminate between promising species. Some of the extracts inhibited the growth of more than one pathogen while other extracts had selective activities and could be the most promising to follow up. The study identified families and orders with either statistically significantly higher or lower antimicrobial activities. Among the large families, Combretaceae and Fabaceae had high mean activities against all test pathogens. The families Anacardiaceae and Moraceae had high activities against both Gram-positive and Gram-negative bacteria whereas the families Proteaceae and Meliaceae had higher antifungal activities. Among the large orders, Fabales had relatively high activities against all the pathogen classes. Considering that plants in related taxa often contain similar compounds and therefore similar activities, future studies could analyse more representative species in the promising taxa. Many tree species, genera, families and orders, including well-known and lesser known medicinal taxa in southern Africa, were identified with promising activities. To evaluate the potential use of these results, additional cytotoxicity, phytochemical and pharmacological studies should be carried out. The study, although still exploratory, underlined the potential of southern African tree extracts as sources of antimicrobial products. Application of these results within the Phytomedicine Programme has led to patents and products that were as good as commercial products in animal and field trials. We hope that our results will provided a starting point for discovering new products with useful activities. / Thesis (PhD)--University of Pretoria, 2014. / gm2014 / Paraclinical Sciences / Unrestricted