Infection process and expression of anti-fungal compounds in avocado (Persea americana Mill.) fruit infected with Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. and molecular identification of fumgal pathogens of avocado

The aims of this project were twofold: (a) to compare the accumulation patterns of antifungal diene (Z,Z)-1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,-15-diene) and triene (Z,Z,E)-1-acetoxy-2-hydroxy-4-oxo-heneicosa-5,12,15-triene) compounds in harvested and unharvested “Fuerte’ avocado fruits in response to inoculation with Colletotrichum gloeosporioides and relate these patterns to observations of the infection cycle, and (b) to use rDNA ITS nucleotide sequence analysis to confirm the identities of some important fungal pathogens of avocado. One of these pathogens was an apparent complex of C. gloeosporioides and Pseudocercospora sp. (CgP complex). Inoculation of harvested and unharvested fruits with C. gloeosporioides at approximately 240 d after fruit set caused an increase in diene and triene levels as measured by HPLC and HPLC-MS compared with controls. In both experiments, these levels reached a maximum during the first 2 d, and subsequently decreased during the 7 d monitoring period. However, there were some notable differences in fluxes of these compounds between experiments. In harvested fruits during the active period, diene levels were higher in the flesh than the peel, whereas triene levels were higher in the peel than the flesh and triene levels decreased to below those of controls in the later stages whereas diene levels did not. Similar patterns emerged for unharvested fruits but diene levels in unharvested fruits were relatively higher than in harvested fruits whereas triene levels were similar in harvested an unharvested fruits. By employing SEM ellipsoid spores characteristic of C. gloeosporioides were first observed on the harvested inoculated fruit surfaces at 2 d, followed by surface hyphae at 3 d. However, the infection process of C. gloeosporioides in unharvested fruits was delayed, relative to harvested fruits, probably due to high antifungal diene and triene contents of unharvested fruits. In contrast to SEM observations, ellipsoid spores of C. gloeosporioides were observed on the inoculated fruit surfaces at 7 d by using CLSM, and no surface hyphae were noted. The ITS universal primers were employed to confirm the identities of the fungal pathogens; Primer pair of ITS5 and ITS4 was used to amplify genomic rDNA from the CgP complex, Pseudocercospora sp. and Phomopsis sp., while ITS1 and ITS4 primers were used to amplify Colletotrichum sp. genomic DNA. Expected PCR product sizes of ~550 base pairs were obtained for all amplifications. Phylogenetic analysis revealed that the CgP sequences were closest to a Phomopsis sp., and confirmed the identity of Pseudocercospora purpurea, Colletotrichum gloeosporioides and Phomopsis perseae

Inhibition of hepatitis B virus replication using synthetic antiviral RNA interference activators

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg 2016Chronic liver infection by hepatitis B virus (HBV) may lead to devastating clinical conditions that include hepatocellular carcinoma and cirrhosis. Approved antiHBV drugs do not completely eradicate the infection, leading to continued viral persistence in infected individuals. Inhibition of HBV replication using synthetic activators of RNA interference (RNAi) may provide a feasible strategy of developing superior antiviral drugs. The aim of this study was to evaluate the therapeutic utility of novel 2’-O-guanidinopropyl (GP) modified synthetic small interfering RNAs (siRNAs) to counter HBV replication in cultured mammalian cells and mice. Initially, single GP moieties were placed at different nucleotide positions of the guide strand of a potent antiHBV siRNA. Some GP-modified siRNAs enhanced antiHBV activity in vitro following transient transfection of Human hepatoma 7 (Huh7) cells with siRNAs and pCH-9/3091, a replication competent HBV target plasmid. These siRNAs inhibited the secretion of Hepatitis B surface antigen (HBsAg) by up to 95% in Huh7 cells. The level of knockdown exhibited by some modified siRNAs was statistically significant relative to that displayed by unmodified siRNA3 which achieved HBsAg silencing of 73%. Additionally, modified siRNAs were also capable of reducing RNA containing the X sequence in vitro by 88-93%. Impressively, some of these knockdown levels were statistically significant when compared to unmodified siRNA3, which achieved HBx knockdown of 83%. Quantitation of interferon (IFN) response genes by reverse transcription quantitative polymerase chain reaction (RTqPCR) and evaluation of cell viability by 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay revealed no evidence of innate immune stimulation or cytotoxicity in cultured cells, respectively. Modified siRNAs also displayed moderate stability in 80% foetal calf serum (FCS). Target specificity was markedly improved by GP-modified siRNAs, especially those with seed modifications (comprising nucleotide position 2 to 8 from the 5’ end of the guide strand). The siRNA-mediated mRNA cleavage product was detected from transfected cells using 5’ Rapid Amplification of cDNA ends (5’ RACE). In the hydrodynamic mouse model, co-injection of GP-modified siRNAs and HBV plasmid vector led to HBsAg suppression of approximately 80-92% at day 3 and 77-96% at day 5 post-administration. The HBV knockdown levels observed at day 3 were statistically significant when compared to those displayed by unmodified siRNA3 which achieved HBsAg silencing of 58% during the same time frame. Furthermore, both sets of siRNAs also suppressed the number of circulating viral particle equivalents (VPEs) by 88- 90% at day 3 post-injection. HBV silencing efficacy of 70-75% and 65% was achieved by modified and unmodified siRNAs, respectively at day 5 post-administration. Finally, antiHBV efficacy of GP-modified siRNAs was tested in HBV transgenic mice following delivery of these RNAi effectors using cationic polyglutamate (PG) adjuvant liposomes. Both groups of antiHBV siRNAs effected HBsAg knockdown that ranged from 70-86% at day 3 to 7 post-administration as siRNA lipoplexes in HBV transgenic mice. In contrast to the unmodified siRNA3, GP-containing siRNAs achieved durable HBsAg silencing of 70% at day 14 post-administration, while the unmodified siRNA3 displayed a shorter duration of activity. As with HBsAg data, the GP-modified siRNAs also displayed silencing efficacy that was similar to the unmodified siRNA, reducing the number of circulating VPEs by 95% from day 3 to 7 post-injection. However, the unmodified siRNA3 lost efficacy by day 14 post-administration, while the GP-modified siRNAs displayed prolonged suppression by reducing the number of circulating VPEs by 75% during the same time interval. Intrahepatic RNA levels were also assessed in transgenic mice, in which GP3 siRNA3 significantly suppressed surface and core RNA levels by 40 and 42%, respectively at day 18 post-injection. The unmodified siRNA3 suppressed surface RNA levels by 20% and core RNA levels by 25% at day 21 post-administration. Furthermore, GP4 siRNA3 silenced both surface and core RNA levels by 42% during the same time period. Additionally, intrahepatic RNA quantitation revealed no induction of IFN response genes by either unmodified or GP-modified siRNAs. In contrast to mice that had received GP-modified siRNAs, significant induction of proinflammatory cytokine release was observed in mice treated with unmodified siRNAs. The siRNA-mediated mRNA cleavage product was also detected from liver samples following 5’ RACE analysis. Neither GPmodified nor unmodified siRNAs significantly induced toxicity in injected mice. Collectively, our data provide evidence that utilisation of GP-modified siRNAs and an efficient hepatotropic non-viral delivery system may be used as a strategy to counter chronic HBV infection.MT201

Lactobacillus rhamnosus cell-free extract targets virulence and antifungal drug resistance in Candida albicans

Candidiasis caused by multidrug-resistant Candida species continues to be difficult to eradicate. The use of live probiotic bacteria has gained a lot of interest in the treatment of candidiasis; however, whole-cell probiotic use can often be associated with a high risk of sepsis. Strategies manipulating cell-free methods using probiotic strains could lead to the development of novel antifungal solutions. Therefore, we evaluated the effect of three probiotic cell-free extracts (CFEs) on the growth, virulence traits, and drug efflux pumps in C. albicans. On the basis of its minimum inhibitory concentration, Lactobacillus rhamnosus was selected and assessed against various virulence traits and drug resistance mechanisms. The results showed that L. rhamnosus CFE significantly inhibited hyphae formation and reduced secretion of proteinases and phospholipases. Moreover, L. rhamnosus inhibited the drug efflux proteins in resistant C. albicans strains thus reversing drug resistance. Gene expression data confirmed downregulation of genes associated with microbial virulence and drug resistance following treatment of C. albicans with L. rhamnosus CFE. Through gas chromatography – mass spectrometry chemical characterization, high contents of oleic acid (24.82%) and myristic acid (13.11%) were observed in this CFE. Collectively, our findings indicate that L. rhamnosus may potentially be used for therapeutic purposes to inhibit C. albicans infections.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author