Browsing by Author "Matlhabe, Ofentse"

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    Evaluating the productivity of selected in vitro culture techniques for the production of a locally isolated entomopathogenic nematode
    (2018) Matlhabe, Ofentse
    Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis have gained interest as biocontrol agents of insect pests due to their ability to search and kill soil dwelling insects. Some members of the Oscheius genus have been shown to have insecticidal abilities as a result of their association with bacteria in the Serratia genus. This has led to the consideration of the Oscheius as an EPN genus. The biocontrol potential serves as an incentive for studying EPNs and various production methods for their commercial use as biopesticides. A putative EPN was isolated from soil samples collected at Brits in the North West Province of South Africa. 18S rDNA based identification indicated that it belonged to the Heterorhabditis genus however, phylogenetic analysis and symptoms on Galleria mellonella larvae suggested that the nematode may belong to the Oscheius genus. The bacterial symbiont associated with this nematode was found to be a strain of Serratia marcescens, through 16S rDNA based sequencing and phylogenetic analysis. Various in vitro production methods were evaluated for their effect in the production of Oscheius L8 MCB. These include monoxenic and axenic culturing, growth media supplementation, and production in solid and liquid state. Axenic cultures were found to produce high maximum yields of EPNs (78 600 EPNs/ml) compared to monoxenic cultures (53 833 EPNs/ml). It was concluded that axenic culturing was efficient for the production of this species. Oscheius L8 MCB was cultured in NB supplemented with varying concentrations of oil and glucose. Supplementation of NB with 2% canola produced a significant amount of EPNs in reduced culture times, but NB supplemented with 4% canola oil and 25mg/ml glucose increased nematode yield but prolonged the culture time. It is noted that media composition (with regards lipid and carbohydrate content) plays an important role in nematode yield and culture time and thus optimization of these components is critical for efficient nematode production. Solid state and liquid state production of Oscheius L8 MCB showed that solid state cultures allowed for early IJ production, whereas liquid state culturing produced the highest IJ yield (36 000 IJs/ml). The reduced culture period, makes solid state production more cost effective and preferable for mass production. However, liquid production can still be used as it offers the benefits of high nematode yeild and efficient recovery of nematodes from culture. The in vitro methods of EPN production have been reported to have an effect on the efficacy of the EPNs against insect hosts. Dose-response assays showed that in vivo produced EPNs resulted in high G. mellonella larvae mortality at lower concentrations compared to solid and liquid in vitro methods. Larvae infected with in vivo produced Oscheius L8 MCB produced a high ix number of emerging IJs compared to larvae infected with EPNs produced using axenic in vitro culturing methods. The differences between mortality and IJ emergence in larvae infected with solid state and liquid state cultured EPNs were marginal. Therefore, it is concluded that axenic culturing methods may reduce the efficacy of Oscheius L8 MCB against insect hosts.
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    Evaluating the productivity of selected in vitro culture techniques for the production of a locally isolated entomopathogenic nematode
    (2018) Matlhabe, Ofentse
    Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis have gained interest as biocontrol agents of insect pests due to their ability to search and kill soil dwelling insects. Some members of the Oscheius genus have been shown to have insecticidal abilities as a result of their association with bacteria in the Serratia genus. This has led to the consideration of the Oscheius as an EPN genus. The biocontrol potential serves as an incentive for studying EPNs and various production methods for their commercial use as biopesticides. A putative EPN was isolated from soil samples collected at Brits in the North West Province of South Africa. 18S rDNA based identification indicated that it belonged to the Heterorhabditis genus however, phylogenetic analysis and symptoms on Galleria mellonella larvae suggested that the nematode may belong to the Oscheius genus. The bacterial symbiont associated with this nematode was found to be a strain of Serratia marcescens, through 16S rDNA based sequencing and phylogenetic analysis. Various in vitro production methods were evaluated for their effect in the production of Oscheius L8 MCB. These include monoxenic and axenic culturing, growth media supplementation, and production in solid and liquid state. Axenic cultures were found to produce high maximum yields of EPNs (78 600 EPNs/ml) compared to monoxenic cultures (53 833 EPNs/ml). It was concluded that axenic culturing was efficient for the production of this species. Oscheius L8 MCB was cultured in NB supplemented with varying concentrations of oil and glucose. Supplementation of NB with 2% canola produced a significant amount of EPNs in reduced culture times, but NB supplemented with 4% canola oil and 25mg/ml glucose increased nematode yield but prolonged the culture time. It is noted that media composition (with regards lipid and carbohydrate content) plays an important role in nematode yield and culture time and thus optimization of these components is critical for efficient nematode production. Solid state and liquid state production of Oscheius L8 MCB showed that solid state cultures allowed for early IJ production, whereas liquid state culturing produced the highest IJ yield (36 000 IJs/ml). The reduced culture period, makes solid state production more cost effective and preferable for mass production. However, liquid production can still be used as it offers the benefits of high nematode yeild and efficient recovery of nematodes from culture. The in vitro methods of EPN production have been reported to have an effect on the efficacy of the EPNs against insect hosts. Dose-response assays showed that in vivo produced EPNs resulted in high G. mellonella larvae mortality at lower concentrations compared to solid and liquid in vitro methods. Larvae infected with in vivo produced Oscheius L8 MCB produced a high number of emerging IJs compared to larvae infected with EPNs produced using axenic in vitro culturing methods. The differences between mortality and IJ emergence in larvae infected with solid state and liquid state cultured EPNs were marginal. Therefore, it is concluded that axenic culturing methods may reduce the efficacy of Oscheius L8 MCB against insect hosts.
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    The characterization of Mycobacterium tuberculosis amidase-like proteins
    (2024) Matlhabe, Ofentse
    Tuberculosis (TB), a disease caused by the bacterium Mycobacterium tuberculosis, is a significant global threat to human health. The emergence of drug resistant M. tuberculosis necessitates the identification of new drug targets for the development of new, shorter regimens. The peptidoglycan (PG) core of the M. tuberculosis cell wall is a potential source of drug targets because it is unique to bacteria and plays a vital role in a multitude of cellular processes and host-mediated immune responses. PG is constantly remodelled by PG synthases and hydrolases in response to external stimuli. This research focuses on N-acetylmuramyl-Lalanine amidases (amidases), PG hydrolases that are implicated in bacterial growth, cell division, virulence and antibiotic tolerance. More specifically, this PhD aims to characterize the M. tuberculosis Ami1 (Rv3717) homologue and to highlight its potential as a drug target. Genotypic characterization of a previously generated M. tuberculosis mutant strain lacking ami1 (H37Δami1S) was conducted prior phenotypic assessments. The deletion of ami1 had no significant effect on growth rate and cell division in standard 7H9 media. In contrast, the growth rate of H37Δami1S was significantly reduced when grown in Sauton’s minimal media with (1%) or without glycerol as a carbon source. We then surmised that Ami1 possibly plays a role in intracellular survival, where host-derived carbon sources support bacterial growth. The survival of H37Δami1S was reduced in IFN-γ stimulated U937 macrophages. H37Δami1S displayed increased susceptibility to rifampicin when assessed by broth microdilution. This observation was credited to a weakened, more permeable cell wall. Consistent with this, H37Δami1S exhibited an increased ethidium bromide uptake. Subsequently, we hypothesized that H37Δami1S may display alterations in antibiotic tolerance/persistence. In a 7-day time-course experiment, H37Δami1S displayed increased susceptibility to vancomycin, ethionamide and isoniazid as evidenced by declining bacterial survival. We interrogated the isoniazid-associated phenotype further, by assessing the transcription of all three amidase-encoding genes. Only ami1 was induced following exposure to isoniazid whereas the expression of ami3 and ami4 remained at basal levels. The regulation of the ami1 gene was explored further through bioinformatics, which revealed two putative transcriptional regulators predicted to bind upstream of ami1, namely Rv1423 and Rv1776c. Protein homology modelling detected HTH DNA binding domains in both proteins. These proteins were then cloned for recombinant expression in the pET29a+ system for purification. Rv1776c was successfully expressed and purified. Electrophoretic mobility shift assays yielded preliminary data that suggested that Rv1776c binds the promoter region of the ami1 gene. Attempts to optimize binding were unsuccessful. To further evaluate the role of Rv1776c and Rv1423 in regulating ami1 gene expression, we over-expressed the regulators, using the tetracycline operator, and assessed effect on cell wall stability, via an ethidium bromide diffusion assay. Over-expression of Rv1776c was not achieved despite increasing concentrations of anhydrotetracycline, suggesting possible downstream regulation of Rv1776c; however, over-expression of Rv1423 was achieved. An increase in ethidium bromide uptake was observed in strains over-expressing Rv1776c and Rv1423. Increasing anhydrotetracycline concentrations in both strains resulted in marginal decreases in the transcription of ami1. Overall, this study has demonstrated that Ami1 plays a vital role in how M. tuberculosis utilizes a carbon source during normal growth and survival in vitro. Moreover, the transcription of ami1 is specifically and directly responsive to isoniazid exposure, possibly via two transcriptional repressors. This work therefore supports further characterization and development of Ami1 as novel drug target in M. tuberculosis.