Browsing by Author "Gessner, Sophia"

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    Catching a glimpse: the visualization of Mycobacterium tuberculosis from TB patient bioaerosols
    (2023) Dinkele, Ryan; Warner, Digby; Gessner, Sophia
    Transmission between hosts is crucial for the success and survival of the obligate human pathogen and aetiological agent of tuberculosis (TB), Mycobacterium tuberculosis (Mtb). Despite this, little is known about how and when Mtb is aerosolized nor the key metabolic and morphological determinants driving successful transmission. To address these knowledge gaps, my doctoral research sought to develop a microscopic method for the detection of aerosolized Mtb following liquidcapture within the respiratory aerosol sampling chamber (RASC). This was achieved through the combination of the mycobacterial cell wall probe, 4-N,Ndimethylamino-1,8-naphthalimide-trehalose (DMN-tre), with the arraying of bioaerosol samples on bespoke nanowell devices amenable to fluorescence microscopy. With this method, a median of 14 live Mtb bacilli (range 0-36) were detected in 90% of confirmed TB patients following 60 minutes of bioaerosol sampling. Three distinct DMN-tre staining patterns were identified among aerosolized Mtb, strongly suggestive of metabolic heterogeneity. Moreover, a low proportion of patients produced Mtb in small clumps. These observations highlight the advantages of using microscopy over conventional culture- or molecular-based techniques for probing the metabolic and morphological characteristics of aerosolized Mtb. Applying this method in a second study, we sought to understand how and when Mtb is aerosolized. To this end, we aimed to compare the aerosolization of Mtb and total particulate matter from patients with TB during three respiratory manoeuvres: tidal breathing (TiBr), forced vital capacity (FVC), and cough. Although total particle counts were 4.8-fold greater in cough samples than either TiBr or FVC, all three manoeuvres returned similar rates of positivity for Mtb. No correlation was observed between total particle production and Mtb count. Instead, for total Mtb counts, the variability between individuals was greater than the variability between sampling manoeuvres. Finally, when modelled using 24-hour breath and cough frequencies, our data indicate that TiBr might contribute more than 90% of the daily aerosolized Mtb among symptomatic TB patients. Assuming the number of viable Mtb organisms detected provides a proxy measure of patient infectiousness, this method suggests that TiBr is a significant contributor to TB transmission. In developing a novel platform for the detection of aerosolized Mtb, this work has suggested the need to re-examine old assumptions about Mtb transmission.
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    Correction to: Bioaerosol sampling of patients with suspected pulmonary tuberculosis: a study protocol
    (2020-08-24) Patterson, Benjamin; Koch, Anastasia; Gessner, Sophia; Dinkele, Ryan; Gqada, Melitta; Bryden, Wayne; Cobelens, Frank; Little, Francesca; Warner, Digby F; Wood, Robin
    An amendment to this paper has been published and can be accessed via the original article
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    Subcellular localization and visualization of RecA and ImuAʹ in mycobacteria
    (2021) Ramudzuli, Atondaho Angelah; Warner, Digby; Gessner, Sophia; Mizrahi, Valerie
    Antibiotic-resistant strains of Mycobacterium tuberculosis (Mtb) are threatening global efforts to eradicate tuberculosis (TB). One attractive approach for target-based drug design proposes to curb the evolution of Mtb during both immune and drug assault. The potential target: mycobacterial DNA metabolism. For this, an in-depth understanding of the mechanisms of DNA repair and mutagenesis in mycobacteria is required. RecA and ImuAʹ are DNA damageinducible proteins implicated in DNA damage repair and tolerance in Mtb. RecA is a key regulatory protein of the SOS response and ImuAʹ is a component of the mycobacterial mutasome, effecting DNA damage tolerance and mutagenesis. In this study, a comprehensive panel of M. smegmatis (Msm) RecA and ImuAʹ reporter strains was generated to explore the dynamics of their expression and subcellular localization within live Msm cells. To this end, fluorescently tagged versions of ImuAʹ and RecA were constructed and shown to retain functional activity in UV-induced mutagenesis but not survival of mitomycin C (MMC) treatment. The discrepant complementation phenotypes observed in UV and MMC assays was unexpected and suggested disruption of a critical protein-protein interaction(s) owing to the presence of the fluorophore. Using fluorescence microscopy, RecA and ImuAʹ expression were monitored in Msm exposed to different types of genotoxic stresses conditions. When mScarletImuAʹ was introduced into wild-type (WT) and ∆imuAʹ backgrounds, diffuse bright red fluorescence was observed in cells treated with MMC and UV; in contrast, no fluorescence expression was observed in untreated cells, confirming the DNA damage-dependent induction of imuAʹ. Following the introduction of RecA-msfGFP into WT and ∆recA backgrounds, discrete green, fluorescent foci were observed in treated and untreated cells in both backgrounds, consistent with the role of RecA in DNA replication in the absence of external DNA damage, and elevated expression under genotoxic conditions. Taken together, these observations support the utility of the fluorescently tagged translational fusions as bioreporters to elucidate the function and regulation of ImuA' and RecA in mycobacteria.