Effects of cigarette smoke condensate on growth and biofilm formation by Mycobacterium tuberculosis

BACKGROUND AND OBJECTIVES : Cigarette smoke (CS) is a major risk factor contributing to the burden of tuberculosis. Little is known, however, about the effects of CS exposure on growth and persistence of Mycobacterium tuberculosis (Mtb) organisms. This issue has been addressed in the current study, which is focused on the effects of cigarette smoke condensate (CSC) on the growth and viability of Mtb planktonic and biofilm-forming cultures. MATERIALS AND METHODS : The planktonic and biofilm-forming cultures were prepared in Middlebrook 7H9 and Sauton broth media, respectively, using Mtb strain, H37Rv. The effects of CSC at concentrations of 0.05-3.12 mg/L on growth, biofilm formation and structure were evaluated using microplate Alamar Blue assay, spectrophotometric procedure and scanning electron microscopy (SEM), respectively. Involvement of reactive oxygen species in CSC-mediated biofilm formation was investigated by including catalase in biofilm-forming cultures. RESULTS : CSC did not affect the growth of planktonic bacteria, but rather led to a statistically significant increase in biofilm formation at concentrations of 0.4-3.12 mg/L, as well as in the viability of biofilm-forming bacteria at CSC concentrations of 0.2-1.56 mg/L. SEM confirmed an agglomerated biofilm matrix and irregular bacterial morphology in CSC-treated biofilms. Inclusion of catalase caused significant attenuation of CSC-mediated augmentation of biofilm formation by Mtb, implying involvement of oxidative stress. These findings demonstrate that exposure of Mtb to CSC resulted in increased biofilm formation that appeared to be mediated, at least in part, by oxidative stress, while no effect on planktonic cultures was observed. CONCLUSION : smoking related augmentation of biofilm formation by Mtb may contribute to persistence of the pathogen, predisposing to disease reactivation and counteracting the efficacy of antimicrobial chemotherapy.South African National Research Foundationhttp://www.hindawi.com/journals/bmripm2020ImmunologyMedical Microbiolog

Utilization of cellulose microcapillary tubes as a model system for culturing and viral infection of mammalian cells

Cryofixation by high-pressure freezing (HPF) and freeze substitution (FS) gives excellent preservation of intracellular membranous structures, ideal for ultrastructural investigations of virus infected cells. Conventional sample preparation methods of tissue cultured cells can however disrupt the association between neighbouring cells or of viruses with the plasma membrane, which impacts upon the effectiveness whereby virus release from cells can be studied. We established a system for virus infection and transmission electron microscopy preparation of mammalian cells that allowed optimal visualisation of membrane release events. African horse sickness virus (AHSV) is a non-enveloped virus that employs two different release mechanisms from mammalian cells, i.e. lytic release through a disrupted plasma membrane and a non-lytic buddingtype release. Cellulose microcapillary tubes were used as support layer for culturing Vero cells. The cells grew to a confluent monolayer along the inside of the tubes and could readily be infected with AHSV. Sections of the microcapillary tubes proved easy to manipulate during the HPF procedure, showed no distortion or compression, and yielded well preserved cells in their native state. There was ample cell surface area available for visualisation, which allowed detection of both types of virus release at the plasma membrane at a significantly higher frequency than when utilising other methods. The consecutive culturing, virus infection and processing of cells within microcapillary tubes therefore represent a novel model system for monitoring intracellular virus life cycle and membrane release events, specifically suited to viruses that do not grow to high titres in tissue culture.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1097-002

Subcellular dynamics studies of iron reveal how tissue‐specific distribution patterns are established in developing wheat grains

Understanding the mechanisms of iron trafficking in plants is key to enhancing the nutritional quality of crops. Because it is difficult to image iron in transit, we currently have an incomplete picture of the route(s) of iron translocation in developing seeds and how the tissue-specific distribution is established. We have used a novel approach, combining iron-57 ( 57Fe) isotope labelling and nanoscale secondary ion mass spectrometry (NanoSIMS), to visualize iron translocation between tissues and within cells in immature wheat grain, Triticum aestivum. This enabled us to track the main route of iron transport from maternal tissues to the embryo through the different cell types. Further evidence for this route was provided by genetically diverting iron into storage vacuoles, with confirmation provided by histological staining and transmission electron microscopy energy dispersive X-ray spectroscopy (TEM-EDS). Almost all iron in both control and transgenic grains was found in intracellular bodies, indicating symplastic rather than apoplastic transport. Furthermore, a new type of iron body, highly enriched in 57Fe, was observed in aleurone cells and may represent iron being delivered to phytate globoids. Correlation of the 57Fe enrichment profiles obtained by NanoSIMS with tissue-specific gene expression provides an updated model of iron homeostasis in cereal grains with relevance for future biofortification strategies

Fungal plant pathogen “mutagenomics” reveals tagged and untagged mutations in Zymoseptoria tritici and identifies SSK2 as key morphogenesis and stress-responsive virulence factor

“Mutagenomics” is the combination of random mutagenesis, phenotypic screening, and whole-genome re-sequencing to uncover all tagged and untagged mutations linked with phenotypic changes in an organism. In this study, we performed a mutagenomics screen on the wheat pathogenic fungus Zymoseptoria tritici for altered morphogenetic switching and stress sensitivity phenotypes using Agrobacterium-mediated “random” T-DNA mutagenesis (ATMT). Biological screening identified four mutants which were strongly reduced in virulence on wheat. Whole genome re-sequencing defined the positions of the T-DNA insertion events and revealed several unlinked mutations potentially affecting gene functions. Remarkably, two independent reduced virulence mutant strains, with similarly altered stress sensitivities and aberrant hyphal growth phenotypes, were found to have a distinct loss of function mutations in the ZtSSK2 MAPKKK gene. One mutant strain had a direct T-DNA insertion affecting the predicted protein’s N-terminus, while the other possessed an unlinked frameshift mutation towards the C-terminus. We used genetic complementation to restore both strains’ wild-type (WT) function (virulence, morphogenesis, and stress response). We demonstrated that ZtSSK2 has a non-redundant function with ZtSTE11 in virulence through the biochemical activation of the stress-activated HOG1 MAPK pathway. Moreover, we present data suggesting that SSK2 has a unique role in activating this pathway in response to specific stresses. Finally, dual RNAseq-based transcriptome profiling of WT and SSK2 mutant strains revealed many HOG1-dependent transcriptional changes in the fungus during early infection and suggested that the host response does not discriminate between WT and mutant strains during this early phase. Together these data define new genes implicated in the virulence of the pathogen and emphasise the importance of a whole genome sequencing step in mutagenomic discovery pipelines

High temperature tolerance in a novel, high-quality phaseolus vulgaris breeding line is due to maintenance of pollen viability and successful germination on the stigma

The common bean (Phaseolus vulgaris L.) is an important nutritional source globally but is sensitive to high temperatures and thus particularly vulnerable to climate change. Derived from a breeding program at CIAT (Colombia), a heat-tolerant breeding line, named heat-tolerant Andean-type 4 (HTA4), was developed by a series of crosses of parents with a small-bean tepary genotype (Phaseolus acutifolius L.) in their pedigree, which might be the donor of heat stress (HS) tolerance. Importantly, in HTA4, the large, commercially desirable Andean-type beans was restored. To assess underlying tolerance mechanisms, HTA4, together with a heat-sensitive Colombian variety (Calima), was exposed to HS (31 °C/24 °C HS vs. 26 °C/19 °C day/night) under controlled environment conditions. Vegetative growth and photosynthetic performance were not negatively impacted by HS in either genotype, although senescence was delayed in Calima. HS during the reproductive stage caused an increase in pod number in Calima but with few fully developed seeds and many pods aborted and/or abscised. In contrast, HTA4 maintained a similar filled pod number under HS and a higher seed weight per plant. Pollen showed high sterility in Calima, with many non-viable pollen grains (24.9% viability compared to 98.4% in control) with a thicker exine and fewer starch granules under HS. Calima pollen failed to adhere to the stigma and germinate under HS. In HTA4, pollen viability was significantly higher than in Calima (71.1% viability compared to 95.4% under control), and pollen successfully germinated and formed pollen tubes in the style under HS. It is concluded that HTA4 is heat tolerant and maintains a high level of reproductive output due to its ability to produce healthy pollen that is able to adhere to the stigma

Targeted mutational analysis to unravel the complexity of African horse sickness virus NS3 function in mammalian cells

The African horse sickness virus non-structural protein 3 (NS3) is involved in the final stages of infection. To gain insight into the function of different NS3 domains, we generated reverse genetics-derived mutants, each expressing a modified version of the protein. A functional comparison of these mutants to the wild-type virus in mammalian cells indicated the variable contribution of the different domains to the cytopathic effect and in ensuring effective virus trafficking and release. The transmembrane domains were determined as essential mediators of NS3 localisation, as the abnormal processing of these mutant proteins resulted in their nuclear localisation and interaction with NS1. NS3 cytoplasmic domain disruptions resulted in increased cytosolic virus particle accumulation and abnormal virion tethering to plasma membranes. Other aspects of infection were also affected, such as VIB formation and distribution of the outer capsid proteins. Overall, these results illustrate the intricate role of NS3 in the infection cycle.The University of Pretoria Institutional Research Theme, South Africa (Grant A0V004) and the Poliomyelitis Research Foundation, South Africa (Grants 13/21 and 16/26). Graduate bursary support was received from the National Research Foundation of South Africa, South Africa (Grant number 102209 and 116412), the Poliomyelitis Research Foundation, South Africa (Grant 14/91) and the University of Pretoria, South Africa.http://www.elsevier.com/locate/yviro2020-05-01hj2019BiochemistryGeneticsMicrobiology and Plant Patholog

Structural (gross and micro), physical and nutritional properties of Trichilia emetica and Trichilia dregeana seeds

This study assessed the gross-structure, micro-structure, physical characteristics and nutritional composition of Trichilia emetica and Trichilia dregeana seeds. T. emetica and T. dregeana seeds have potential for commercialization and improved food security, yet they are under-utilized and under-researched. The gross- and micro-structure of the seeds was assessed using stereo microscopy, light microscopy (LM), confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Protein, fat, ash, and mineral content of the seeds were also assessed. T. emetica and T. dregeana seeds had lower bulk density (393.5 kgm−3 & 433.6 kgm−3 respectively) and lower porosity (55.07% & 54.38% respectively) than soybean. Geometric mean diameter (29.7 mm & 16.9 mm) and aspect ratio (1.72 and 1.85) of T. emetica and T. dregeana seeds respectively, were higher than soybean, while they had lower sphericity (0.66 and 0.66 respectively) than soybean (0.99). Microscopy analysis showed that Trichilia species had larger-sized and more round-shaped protein bodies than soybean seeds. T. emetica and T. dregeana protein contents (25.6% and 17.3% w/w, respectively) were lower than soybean (45.4%) while fat contents (49% and 51.5% w/w, respectively) were higher than soybean (20.2% w/w). Potassium (1075–1350 mg100g−1) and calcium (285–300 mg100g−1) where the main macro-minerals while iron (6.33–6.83 mg100g−1) and zinc (2.30–2.90 mg100g−1) were the main micro-minerals. The research demonstrated that the structural and nutritional characteristics of T. dregeana and T. emetica seeds could facilitate their commercial utilization and application for food security alleviation.Este estudio evaluó la estructura bruta, la microestructura, las características físicas y la composición nutricional de las semillas de Trichilia emetica y Trichilia dregeana. Si bien las semillas de T. emetica y T. dregeana poseen potencial para ser comercializadas y mejorar la seguridad alimentaria, son infrautilizadas y han sido poco investigadas. Para evaluar algunos de los aspectos mencionados —estructura bruta y microestructura— de las semillas se emplearon microscopía estereoscópica, microscopía de luz (LM), microscopía confocal de barrido láser (CLSM) y microscopía electrónica de barrido (SEM). Además, se evaluó el contenido de proteínas, grasas, cenizas y minerales de las mismas. Se constató que las semillas de T. emetica y T. dregeana tienen una densidad aparente menor (393.5 kgm−3 y 433.6 kgm−3, respectivamente) y una porosidad menor (55.07% y 54.38%, respectivamente) que las de la soya. El diámetro geométrico medio (29.7 mm y 16.9 mm) y la ratio de aspecto (1.72 y 1.85) de las semillas de T. emetica y T. dregeana, respectivamente, son mayores que los de la soya, mientras que su esfericidad es menor (0.66 y 0.66, respectivamente) que la de la soya (0.99). El análisis microscópico permitió comprobar que las especies de Trichilia poseen cuerpos proteicos de mayor tamaño que las semillas de soya y que su forma es redonda. Asimismo, se constató que los contenidos proteicos de T. emetica y T. dregeana (25.6% y 17.3% p/p, respectivamente) son inferiores a los de la soya (45.4%), mientras que los contenidos grasos (49% y 51.5% p/p, respectivamente) superan los de la soya (20.2% p/p). Los principales macrominerales detectados son potasio (1075-1350 mg100g−1) y calcio (285-300 mg100g−1), y los principales microminerales hierro (6.33-6.83 mg100g−1) y zinc (2.30-2.90 mg100g−1). La investigación dio cuenta de que las características estructurales y nutricionales de las semillas de T. dregeana y T. emetica podrían facilitar su utilización comercial y su aplicación para garantizar la seguridad alimentaria.National Research Foundation (NRF), Agricultural Research Council (ARC) PDP Programme, National Research Foundation South Africa.http://www.tandfonline.com/toc/tcyt20/currentpm2021Genetic