Browsing by Author "Kunota, Tafara Takunda Remigio."

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Hydrogen sulfide (H2S) production by mycobacteria.
(2020) Kunota, Tafara Takunda Remigio.; Steyn, Andrie J. C.
The gasotransmitter hydrogen sulfide (H2S) has been recognized as a physiological mediator with a variety of functions across all domains of life. Many prokaryotic bacterial species endogenously generate H2S in their natural environments. However, to date, it is not known whether Mycobacterium tuberculosis (Mtb) is an endogenous producer of H2S. In this study, we tested the hypothesis that Mtb endogenously produces H2S to modulate respiration, central metabolism, oxidative stress, and drug susceptibility. We demonstrated that fast-growing non-pathogenic, slow-growing pathogenic mycobacterial species, as well as drug resistant clinical strains of Mtb species produce H2S. Here we demonstrate that fast-growing non-pathogenic M. smegmatis produces barely detectable quantities of H2S, whereas MDR Mtb produces large quantities of H2S. We have also developed a native PAGEbased assay for the rapid screening of H2S producing enzymes in the lysates of mycobacterial species. Using LC-MS/MS, we identified the protein, Rv3684 as an H2S-producing enzyme in Mtb. Disruption of rv3684, demonstrated using the genetic knock out of rv3684, reduces, but does not eliminate, H2S production, suggesting the involvement of multiple genes in H2S production. Whole Mtb cell-based and lysate assays showed reduced levels of H2S production in the Mtb knockout strain compared to the wild-type strain. Noticeably, we demonstrated that the Mtb mutant is resistant to oxidative stress and the anti-TB drugs clofazimine and rifampicin. We also found that endogenous H2S is an effector molecule that maintains bioenergetic homeostasis by regulating Mtb respiration, and that H2S also plays a key role in central metabolism by modulating the balance between oxidative phosphorylation (OXPHOS) and glycolysis. In summary, our findings reveal previously unknown concepts of Mtb physiology with respect to Mtb-derived H2S and energy metabolism which has significant implication for routine laboratory culturing, understanding susceptibility and TB disease.
Technologies for a user-friendly microfluidic system for portable applications.
(2015) Kunota, Tafara Takunda Remigio.; Balagaddé, Frederick.
In the same way that the HIV virus subdues the human immune system, the HIV/AIDS epidemic has severely overloaded the health service infrastructure in resource limited countries and threatens to systematically suppress societies’ capacity to cope with killer diseases. The epidemic has also directly impacted the health workforce, causing absenteeism, attrition (due to illness and death), and increased demand for provider time and skills. Advanced and miniaturized microfluidic systems can perform complex biotechnological functions such as growing bacteria, sequencing DNA and identifying disease causing pathogens. As a technology, microfluidics offers so many advantages but it also suffers from a variety of technological drawbacks that limit its wide spread practical application in hospitals and patient setting. Microfluidic systems require a lot of time (6 hours to an entire work-day) to set up and the set-up process requires the meticulous attention of highly trained personnel. We proposed the development of an automated, time conservative and user-friendly fluid-transport system (off-chip to on-chip) for Microfluidic Large Scale Integration platform based microfluidic devices. Using multilayer soft-lithography, micro-electric actuators and a LabVIEW graphical user-interface, a user-friendly automated microfluidic fluid transport system was developed. In comparison to the conventional manual loading system, the developed system can save at least 60% of the total chip preparation time required during the off-chip to on-chip fluid loading process. This system can be extended and made compatible with other devices that require complex off-chip to on-chip loading processes in microfluidic large scale integration platform based systems.