Thesis (Ph.D.(Microbiology)) -- University of Limpopo, 2022 / Respiratory tract infections (RTIs) are frequent ailments among humans and are a
high burden to public health. One strategy for the development of new therapies
against pathogenic bacteria such as Mycobacterium tuberculosis is to target essential
biosynthetic pathways of its metabolism. The aim of this study was to evaluate and
target the biosynthesis of aromatic amino acids (shikimate pathway) of Mycobacterial
spp using medicinal plant extracts. The selection of the plants in this study was based
on their ethnopharmacological use for the treatment of tuberculosis infections and
related symptoms. The leaves were dried at ambient temperatures and ground to fine
powder. The powdered material was extracted with hexane, dichloromethane,
acetone, methanol and water. Phytochemical screening was done using standard
protocols that tested for tannins, saponins, terpenoids, alkaloids, flavonoids, steroids,
anthraquinones, phlobatannins, quinones, and betacynins. Phytochemical fingerprints
were established using thin layer chromatography (TLC) where three mobile phases
varying in polarity were used to develop the chromatograms. Total Phenolics,
flavonoids, flavonols, tannins, alkaloids and proanthocyanidin contents were
quantified using UV/Vis spectrometry. Spectrometric quantification of the free radical
(DPPH) scavenging activity and ferric (potassium ferricyanide) reducing power were
performed. The heat-dependent bovine serum albumin and egg albumin denaturation
assays were used to evaluate anti-inflammatory activity. Antimycobacterial activity
was screened using bioautography assay in qualitative analysis. Quantitatively, broth
microdilution assay was used to determine the minimal inhibitory concentrations. The
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9
interference genetic editing technique was used to evaluate and validate the
essentiality of the aromatic amino acids in Mycobacteria to further determine the
vulnerability and draggability of the transketolase (tkt) and DAHPs (aroG) genes.
Plasmid, PLJR962, was used for the CRISPRi/dCas9 gene knockdown experiments.
The integrating CRISPRi plasmid expressed both sgRNA with the targeting region (for
tkt or aroG) and the dCas9 handle which is under control of the anhydrotetracycline
(ATC) inducible promoters. The spot assay and growth curves were used to for
phenotypic characterisation and gene knockdown experiments. RNA microarray
(qPCR) was used to evaluate the level of expression inhibition of tkt gene . Mechanism
of action of plants extracts bioactive components were predicted based on synergy
between gene knockdown, shikimate inhibitors and the plant extracts. To evaluate
whether the shikimate intermediates may rescue gene depleted M. smegmatis
hypomorphs, the cultures were grown in L-tryptophan, L-phenylalanine, L-tyrosine and
shikimic acid and growth curves constructed. Cytotoxicity of the extracts was
evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay on Vero cell lines and phorbol 12-myristate 13-acetate (PMA)
differentiated THP-1 macrophages. Phytochemical analysis showed that the various
extracts had various polar and non-polar compounds which belonged to phenolics,
saponins, steroids, terpenoids, alkaloids, cardiac glycosides and resins. Numerous
non-polar compounds from Gardernia volkensii, Senna petersiana, Ficus sur had
antimycobacterial activity against M. smegmatis in bioautography. Remarkably,
acetone extracts from S. petersiana, Acacia senegal, Carissa bispinosa, P. africanum
and C. gratissimus that had moderate to low antimycobacterial activity against wild type M. smegmatis (mc2 155) demonstrated improved inhibitory activity against the tkt
PAM1 M. smegmatis CRISPRi mutant. Only the acetone Clerodendrum glabrum,
Croton gratissimus, Peltophorum africanum and Gardenia volkensii demonstrated
activity against M. tuberculosis H37Rv. These results suggest that the employment of
CRISPRi in M. tuberculosis to develop screening models may increase changes of
obtaining bioactive chemical species because the tkt gene knockdown was showed to
possess the ability to potentiate the antimycobacterial activity of the plant extracts. An
added advantage of the plant extracts is their antioxidant and anti-inflammatory
activities which may benefit the host immune system during treatment of infection by
reducing free radicals and pro-inflammatory agents that perpetuate the infection. Non polar compounds were found to generally have higher anti-inflammatory activity than
the polar counterpart for all the plant extracts. These results suggest that the non-polar
compounds from the tested extracts may not only confer antimycobacterial effects, but
also anti-inflammatory activities. A. senegal, G. volkensii, F. sur, S. petersiana and C.
glabrum were found to be toxic to the Vero cell line. However, purification techniques
may circumvent their toxic effects. This study demonstrated that the amino acid
biosynthesis is a potential antimycobacterial drug target because it was found to be
essential, vulnerable and druggable by medicinal plant extracts / University of Limpopo and
National Research Foundation
(NRF-DAAD In-Country Doctoral Scholarship Programme)
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ul/oai:ulspace.ul.ac.za:10386/4151 |
| Date | January 2022 |
| Creators | Matotoka, Mashilo Mash |
| Contributors | Masoko, P., Mashabela, G. T. B. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | xxiv, 211 leaves |
| Relation |
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