Department of Chemistry and Biochemistry

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Open Access
AFM-based correlative microscopy illuminates human pathogens
(Frontiers Media, 2021-05-07) Bhat, Supriya V.; Price, Jared D. W.; Dahms, Tanya E. S.
Microbes have an arsenal of virulence factors that contribute to their pathogenicity. A number of challenges remain to fully understand disease transmission, fitness landscape, antimicrobial resistance and host heterogeneity. A variety of tools have been used to address diverse aspects of pathogenicity, from molecular host-pathogen interactions to the mechanisms of disease acquisition and transmission. Current gaps in our knowledge include a more direct understanding of host-pathogen interactions, including signaling at interfaces, and direct phenotypic confirmation of pathogenicity. Correlative microscopy has been gaining traction to address the many challenges currently faced in biomedicine, in particular the combination of optical and atomic force microscopy (AFM). AFM, generates high-resolution surface topographical images, and quantifies mechanical properties at the pN scale under physiologically relevant conditions. When combined with optical microscopy, AFM probes pathogen surfaces and their physical and molecular interaction with host cells, while the various modes of optical microscopy view internal cellular responses of the pathogen and host. Here we review the most recent advances in our understanding of pathogens, recent applications of AFM to the field, how correlative AFM-optical microspectroscopy and microscopy have been used to illuminate pathogenicity and how these methods can reach their full potential for studying host- pathogen interactions.
Open Access
Altered Envelope Structure and Nanomechanical Properties of a C-Terminal Protease A-Deficient Rhizobium leguminosarum
(Multidisciplinary Digital Publishing Institute, 2020-09-16) Jun, Dong; Idem, Ubong; Dahms, Tanya E. S.
1) Background: Many factors can impact bacterial mechanical properties, which play an important role in survival and adaptation. This study characterizes the ultrastructural phenotype, elastic and viscoelastic properties of Rhizobium leguminosarum bv. viciae 3841 and the C-terminal protease A (ctpA) null mutant strain predicted to have a compromised cell envelope; (2) Methods: To probe the cell envelope, we used transmission electron microscopy (TEM), high performance liquid chromatography (HPLC), mass spectrometry (MS), atomic force microscopy (AFM) force spectroscopy, and time-dependent AFM creep deformation; (3) Results: TEM images show a compromised and often detached outer membrane for the ctpA mutant. Muropeptide characterization by HPLC and MS showed an increase in peptidoglycan dimeric peptide (GlcNAc-MurNAc-Ala-Glu-meso-DAP-Ala-meso-DAP-Glu-Ala-MurNAc-GlcNAc) for the ctpA mutant, indicative of increased crosslinking. The ctpA mutant had significantly larger spring constants than wild type under all hydrated conditions, attributable to more highly crosslinked peptidoglycan. Time-dependent AFM creep deformation for both the wild type and ctpA mutant was indicative of a viscoelastic cell envelope, with best fit to the four-element Burgers model and generating values for viscoelastic parameters k1, k2, η1, and η2; (4) Conclusions: The viscoelastic response of the ctpA mutant is consistent with both its compromised outer membrane (TEM) and fortified peptidoglycan layer (HPLC/MS).
Open Access
Back to Nature: Combating Candida albicans Biofilm, Phospholipase and Hemolysin Using Plant Essential Oils
(Multidisciplinary Digital Publishing Institute, 2021-01-15) El-Baz, Ahmed M.; Mosbah, Rasha A.; Goda, Reham M.; Mansour, Basem; Sultana, Taranum; Dahms, Tanya E. S.; El-Ganiny, Amira M.
Candida albicans is the causative agent of fatal systemic candidiasis. Due to limitations of antifungals, new drugs are needed. The anti-virulence effect of plant essential oils (EOs) was evaluated against clinical C. albicans isolates including cinnamon, clove, jasmine and rosemary oils. Biofilm, phospholipase and hemolysin were assessed phenotypically. EOs were evaluated for their anti-virulence activity using phenotypic methods as well as scanning electron microscopy (SEM) and atomic force microscopy (AFM). Among the C. albicans isolates, biofilm, phospholipase and hemolysins were detected in 40.4, 86.5 and 78.8% of isolates, respectively. Jasmine oil showed the highest anti-biofilm activity followed by cinnamon, clove and rosemary oils. SEM and AFM analysis showed reduced adherence and roughness in the presence of EOs. For phospholipase, rosemary oil was the most inhibitory, followed by jasmine, cinnamon and clove oils, and for hemolysins, cinnamon had the highest inhibition followed by jasmine, rosemary and clove oils. A molecular docking study revealed major EO constituents as promising inhibitors of the Als3 adhesive protein, with the highest binding for eugenol, followed by 1,8-cineole, 2-phenylthiolane and cinnamaldehyde. In conclusion, EOs have a promising inhibitory impact on Candida biofilm, phospholipase and hemolysin production, hence EOs could be used as potential antifungals that impact virulence factors.
Open Access
Candida albicans Reactive Oxygen Species (ROS)-Dependent Lethality and ROS-Independent Hyphal and Biofilm Inhibition by Eugenol and Citral.
(American Society for Microbiology, 2022-11-17) Shahina, Zinnat; Ndlovu, Easter; Persaud, Omkaar; Sultana, Taranum; Dahms, Tanya E. S.
Candida albicans is part of the normal human flora but is most frequently isolated as the causative opportunistic pathogen of candidiasis. Plant-based essential oils and their components have been extensively studied as antimicrobials, but their antimicrobial impacts are poorly understood. Phenylpropenoids and monoterpenes, for example, eugenol from clove and citral from lemon grass, are potent antifungals against a wide range of pathogens. We report the cellular response of C. albicans to eugenol and citral, alone and combined, using biochemical and microscopic assays. The MICs of eugenol and citral were 1,000 and 256 μg/mL, respectively, with the two exhibiting additive effects based on a fractional inhibitory concentration index of 0.83 ± 0.14. High concentrations of eugenol caused membrane damage, oxidative stress, vacuole segregation, microtubule dysfunction and cell cycle arrest at the G1/S phase, and while citral had similar impacts, they were reactive oxygen species (ROS) independent. At sublethal concentrations (1/2 to 1/4 MIC), both oils disrupted microtubules and hyphal and biofilm formation in an ROS-independent manner. While both compounds disrupt the cell membrane, eugenol had a greater impact on membrane dysfunction. This study shows that eugenol and citral can induce vacuole and microtubule dysfunction, along with the inhibition of hyphal and biofilm formation.
Open Access
Cinnamomum zeylanicum bark essential oil induces cell wall remodelling and spindle defects in Candida albicans
(BMC, 2018-02-09) Shahina, Zinnat; El‑Ganiny, Amira M.; Minion, Jessica; Whiteway, Malcolm; Sultana, Taranum; Dahms, Tanya E. S.
Cinnamon (Cinnamomum zeylanicum) bark extract exhibits potent inhibitory activity against Candida albicans but the antifungal mechanisms of this essential oil remain largely unexplored.
Open Access
Correlative atomic force microscopy quantitative imaging- laser scanning confocal microscopy quantifies the impact of stressors on live cells in real-time
(Nature Research, 2018-05-29) Bhat, Supriya V.; Sultana, Taranum; Körnig, André; McGrath, Seamus; Shahina, Zinnat; Dahms, Tanya E. S.
There is an urgent need to assess the effect of anthropogenic chemicals on model cells prior to their release, helping to predict their potential impact on the environment and human health. Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) have each provided an abundance of information on cell physiology. In addition to determining surface architecture, AFM in quantitative imaging (QI) mode probes surface biochemistry and cellular mechanics using minimal applied force, while LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Correlative AFM-LSCM produces complimentary information on different cellular characteristics for a comprehensive picture of cellular behaviour. We present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ, producing multiplexed data on cell morphology and mechanics, surface adhesion and ultrastructure, and real-time localization of multiple fluorescently tagged macromolecules. To demonstrate the broad applicability of this method for disparate cell types, we show altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. AFM-QI-LSCM is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.
Open Access
EF1025, a hypothetical protein from Enterococcus faecalis, interacts with DivIVA and affects cell length and cell shape
(Frontiers Media, 2020-02-12) Sharma, Kusum; Sultana, Taranum; Liao, Mingmin; Dahms, Tanya E. S.; Dillon, Jo-Anne R.
DivIVA plays multifaceted roles in Gram-positive organisms through its association with various cell division and non-cell division proteins. We report a novel DivIVA interacting protein in Enterococcus faecalis, named EF1025 (encoded by EF1025), which is conserved in Gram-positive bacteria. The interaction of EF1025 with DivIVAEf was confirmed by Bacterial Two-Hybrid, Glutathione S-Transferase pull-down, and co-immunoprecipitation assays. EF1025, which contains a DNA binding domain and two Cystathionine β-Synthase (CBS) domains, forms a decamer mediated by the two CBS domains. Viable cells were recovered after insertional inactivation or deletion of EF1025 only through complementation of EF1025 in trans. These cells were longer than the average length of E. faecalis cells and had distorted shapes. Overexpression of EF1025 also resulted in cell elongation. Immuno-staining revealed comparable localization patterns of EF1025 and DivIVAEf in the later stages of division in E. faecalis cells. In summary, EF1025 is a novel DivIVA interacting protein influencing cell length and morphology in E. faecalis.
Open Access
Exposure to sub-lethal 2,4-dichlorophenoxyacetic acid arrests cell division and alters cell surface properties in Escherichia coli
(Frontiers Media, 2018-02-01) Bhat, Supriya V.; Kamencic, Belma; Körnig, André; Shahina, Zinnat; Dahms, Tanya E. S.
Escherichia coli is a robust, easily adaptable and culturable bacterium in vitro, and a model bacterium for studying the impact of xenobiotics in the environment. We have used correlative atomic force – laser scanning confocal microscopy (AFM-LSCM) to characterize the mechanisms of cellular response to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). One of the most extensively used herbicides world-wide, 2,4-D is known to cause hazardous effects in diverse non-target organisms. Sub-lethal concentrations of 2,4-D caused DNA damage in E. coli WM1074 during short exposure periods which increased significantly over time. In response to 2,4-D, FtsZ and FtsA relocalized within seconds, coinciding with the complete inhibition of cell septation and cell elongation. Exposure to 2,4-D also resulted in increased activation of the SOS response. Changes to cell division were accompanied by concomitant changes to surface roughness, elasticity and adhesion in a time-dependent manner. This is the first study describing the mechanistic details of 2,4-D at sub-lethal levels in bacteria. Our study suggests that 2,4-D arrests E. coli cell division within seconds after exposure by disrupting the divisome complex, facilitated by dissipation of membrane potential. Over longer exposures, 2,4-D causes filamentation as a result of an SOS response to oxidative stress induced DNA damage.
Open Access
Key essential oil components delocalize Candida albicans Kar3p and impact microtubule structure
(Elsevier, 2023-04-03) Shahina, Zinnat; Yennamalli, Ragothaman M.; Dahms, Tanya E. S.
Treatment of Candida albicans associated infections is often ineffective in the light of resistance, with an urgent need to discover novel antimicrobials. Fungicides require high specificity and can contribute to antifungal resistance, so inhibition of fungal virulence factors is a good strategy for developing new antifungals.
Open Access
Metabolic adaptation of C-terminal protease A-deficient R. leguminosarum in response to loss of nutrient transport
(Frontiers Media, 2018-01-04) Jun, Dong; Minic, Zoran; Bhat, Supriya V.; Vanderlinde, Elizabeth M.; Yost, Chris K.; Babu, Mohan; Dahms, Tanya E. S.
Post-translational modification expands the functionality of the proteome beyond genetic encoding, impacting many cellular processes. Cleavage of the carboxyl terminus is one of the many different ways proteins can be modified for functionality. Gel-electrophoresis and mass spectrometric-based techniques were used to identify proteins impacted by deficiency of a C-terminal protease, CtpA, in Rhizobium leguminosarum bv. viciae 3841. Predicted CtpA substrates from 2D silver stained gels were predominantly outer membrane and transport proteins. Proteins with altered abundance in the wild type and ctpA (RL4692) mutant, separated by 2D difference gel electrophoresis, were selected for analysis by mass spectrometry. Of those identified, 9 were the periplasmic solute-binding components of ABC transporters, 5 were amino acid metabolic enzymes, 2 were proteins involved in sulfur metabolism, and 1 each was related to carbon metabolism, protein folding and signal transduction. Alterations to ABC-binding-cassette transporters, nutrient uptake efficiency and to amino acid metabolism indicated an impact on amino acid metabolism and transport for the ctpA mutant, which was validated by measured amino acid levels.
Open Access
Rhizobium leguminosarum bv. viciae 3841 Adapts to 2,4-Dichlorophenoxyacetic Acid with “Auxin-Like” Morphological Changes, Cell Envelope Remodeling and Upregulation of Central Metabolic Pathways
(Public Library of Science, 2015-04-28) Bhat, Supriya V.; Booth, Sean C.; McGrath, Seamus G. K.; Dahms, Tanya E. S.
There is a growing need to characterize the effects of environmental stressors at the molec- ular level on model organisms with the ever increasing number and variety of anthropogenic chemical pollutants. The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), as one of the most widely applied pesticides in the world, is one such example. This herbicide is known to have non-targeted undesirable effects on humans, animals and soil microbes, but specific molecular targets at sublethal levels are unknown. In this study, we have used Rhizobium leguminosarum bv. viciae 3841 (Rlv) as a nitrogen fixing, beneficial model soil organism to characterize the effects of 2,4-D. Using metabolomics and advanced microscopy we deter- mined specific target pathways in the Rlv metabolic network and consequent changes to its phenotype, surface ultrastructure, and physical properties during sublethal 2,4-D exposure. Auxin and 2,4-D, its structural analogue, showed common morphological changes in vitro- which were similar to bacteroids isolated from plant nodules, implying that these changes are related to bacteroid differentiation required for nitrogen fixation. Rlv showed remarkable adaptation capabilities in response to the herbicide, with changes to integral pathways of cellular metabolism and the potential to assimilate 2,4-D with consequent changes to its physical and structural properties. This study identifies biomarkers of 2,4-D in Rlv and offers valuable insights into the mode-of-action of 2,4-D in soil bacteria.
Open Access
Rosemary essential oil and its components 1,8-cineole and α-pinene induce ROS-dependent lethality and ROS-independent virulence inhibition in Candida albican
(Public Library of Science, 2022-11-16) Shahina, Zinnat; Al Homsi, Raymond; Price, Jared D. W.; Whiteway, Malcolm; Sultana, Taranum; Dahms, Tanya E. S.
The essential oil from Rosmarinus officinalis L., a composite mixture of plant-derived secondary metabolites, exhibits antifungal activity against virulent candidal species. Here we report the impact of rosemary oil and two of its components, the monoterpene α-pinene and the monoterpenoid 1,8-cineole, against Candida albicans, which induce ROS-dependent cell death at high concentrations and inhibit hyphal morphogenesis and biofilm formation at lower concentrations. The minimum inhibitory concentrations (100% inhibition) for both rosemary oil and 1,8-cineole were 4500 μg/ml and 3125 μg/ml for α-pinene, with the two components exhibiting partial synergy (FICI = 0.55 ± 0.07). At MIC and 1/2 MIC, rosemary oil and its components induced a generalized cell wall stress response, causing damage to cellular and organelle membranes, along with elevated chitin production and increased cell surface adhesion and elasticity, leading to complete vacuolar segregation, mitochondrial depolarization, elevated reactive oxygen species, microtubule dysfunction, and cell cycle arrest mainly at the G1/S phase, consequently triggering cell death. Interestingly, the same oils at lower fractional MIC (1/8-1/4) inhibited virulence traits, including reduction of mycelium (up to 2-fold) and biofilm (up to 4-fold) formation, through a ROS-independent mechanism.

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